Formulation of a mixture of free-B-ring flavonoids and flavans as a therapeutic agent

ABSTRACT

The present invention provides a novel composition of matter comprised of a mixture of two specific classes of compounds—Free-B-ring flavonoids and flavans—for use in the prevention and treatment of diseases and conditions mediated by the COX-2 and 5-LO pathways. The present invention further provides a novel method for simultaneously inhibiting the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) enzymes, and reducing cox-2 mRNA production. Finally, the present invention includes a method for weight loss and blood glucose control. The methods of this invention are comprised of administering to a host in need thereof an effective amount of the composition of this invention together with a pharmaceutically acceptable carrier. This invention relates generally to the prevention and treatment of diseases and conditions mediated by the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) pathways, including but not limited to the relief joint discomfort and pain associated with conditions such as osteoarthritis, rheumatoid arthritis, and other injuries that result from overuse.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/427,746, filed Apr. 30, 2003, entitled Formulation Of A Mixture OfFree-B-Ring Flavonoids And Flavans As A Therapeutic Agent, whichapplication claims priority to U.S. Provisional Application Ser. No.60/377,168, filed Apr. 30, 2002, entitled Formulation With Dual COX-2And 5-Lipoxygenase Inhibitory Activity. Each of these references ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the prevention and treatment ofdiseases and conditions mediated by the cyclooxygenase-2 (COX-2) and5-lipoxygenase (5-LO) pathways. Specifically, the present inventionrelates to a novel composition of matter comprised of a mixture of ablend of two specific classes of compounds—Free-B-ring flavonoids andflavans—for use in the prevention and treatment of diseases andconditions mediated by the COX-2 and 5-LO pathways. Included in thepresent invention is a method for the simultaneous inhibition of theprotein function of the COX-2 and 5-LO enzymes, and a method formodulating the production of mRNA by administration of the novelcomposition of this invention. Also included in the present invention isa method for the prevention and treatment of COX-2 and 5-LO mediateddiseases and conditions, including but not limited to joint discomfortand pain associated with conditions such as osteoarthritis, rheumatoidarthritis, and other injuries that result from overuse. Further includedin the present invention is a method for reducing blood glucose levelsand promoting weight loss.

BACKGROUND OF THE INVENTION

The liberation and metabolism of arachidonic acid (AA) from the cellmembrane results in the generation of pro-inflammatory metabolites byseveral different pathways. Arguably, two of the most important pathwaysto inflammation are mediated by the enzymes 5-lipoxygenase (5-LO) andcyclooxygenase (COX). These parallel pathways result in the generationof leukotrienes and prostaglandins, respectively, which play importantroles in the initiation and progression of the inflammatory response.These vasoactive compounds are chemotaxins, which promote infiltrationof inflammatory cells into tissues and serve to prolong the inflammatoryresponse. Consequently, the enzymes responsible for generating thesemediators of inflammation have become the targets for many new drugsaimed at the treatment of inflammation that contributes to thepathogenesis of diseases such as rheumatoid arthritis, osteoarthritis,Alzheimer's disease and certain types of cancer.

Inhibition of the cyclooxygenase (COX) enzyme is the mechanism of actionattributed to most nonsteroidal anti-inflammatory drugs (NSAIDS). Thereare two distinct isoforms of the COX enzyme (COX-1 and COX-2) that shareapproximately 60% sequence homology, but differ in expression profilesand function. COX-1 is a constitutive form of the enzyme that has beenlinked to the production of physiologically important prostaglandinsinvolved in the regulation of normal physiological functions such asplatelet aggregation, protection of cell function in the stomach andmaintenance of normal kidney function (Dannhardt and Kiefer (2001) Eur.J. Med. Chem. 36:109-26). The second isoform, COX-2, is a form of theenzyme that is inducible by pro-inflammatory cytokines such asinterleukin-1β (IL-1β) and other growth factors (Herschmann (1994)Cancer Metastasis Rev. 134:241-56; Xie et al. (1992) Drugs Dev. Res.25:249-65). This isoform catalyzes the production of prostaglandin E₂(PGE₂) from AA. Inhibition of COX-2 is responsible for theanti-inflammatory activities of conventional NSAIDs.

Inhibitors that demonstrate dual specificity for COX-2 and 5-LO, whilemaintaining COX-2 selectivity relative to COX-1, would have the obviousbenefit of inhibiting multiple pathways of AA metabolism. Suchinhibitors would block the inflammatory effects of PGE₂, as well as,those of multiple leukotrienes (LT) by limiting their production. Thisincludes the vasodilation, vasopermeability and chemotactic effects ofLTB₄ and LTD₄ and the effects of LTE₄, also known as the slow reactingsubstance of anaphalaxis. Of these, LTB₄ has the most potent chemotacticand chemokinetic effects (Moore (1985) in Prostanoids: Pharmacological,Physiological and Clinical Relevance, Cambridge University Press, N.Y.,pp. 229-30) and has been shown to be elevated in the gastrointestinalmucosa of patients with inflammatory bowel disease (Sharon and Stenson(1983) Gastroenterology 84:1306-13) and within the synovial fluid ofpatients with rheumatoid arthritis (Klicksein et al. (1980) J. Clin.Invest. 66:1166-70; Rae et al. (1982) Lancet ii:1122-4).

In addition to the above-mentioned benefits of dual COX-2/5-LOinhibitors, many dual inhibitors do not cause some of the side effectsthat are typical of NSAIDs or COX-2 inhibitors, including thegastrointestinal damage and discomfort caused by traditional NSAIDs. Ithas been suggested that NSAID-induced gastric inflammation is largelydue to metabolites of 5-LO, particularly LTB₄, which attracts cells tothe site of a gastric lesion thus causing further damage (Kircher et al.(1997) Prostaglandins Leukot. Essent. Fatty Acids 56:417-23).Leukotrienes represent the primary AA metabolites within the gastricmucosa following prostanoid inhibition. It appears that these compoundscontribute to a significant amount of the gastric epithelial injuryresulting from the use of NSAIDs. (Celotti and Laufer (2001) Pharmacol.Res. 43:429-36). Dual inhibitors of COX-2 and 5-LO were alsodemonstrated to inhibit the coronary vasoconstriction in arthritichearts in a rat model (Gok et al. (2000) Pharmacology 60:41-46). Takentogether, these characteristics suggest that there may be distinctadvantages to dual inhibitors of COX-2 and 5-LO over specific COX-2inhibitors and non-specific NSAIDs with regard to both increasedefficacy and reduced side effects.

Because the mechanism of action of COX inhibitors overlaps that of mostconventional NSAIDs, COX inhibitors are used to treat many of the samesymptoms, such as the pain and swelling associated with inflammation intransient conditions and chronic diseases in which inflammation plays acritical role. Transient conditions include the treatment ofinflammation associated with minor abrasions, sunburn or contactdermatitis, as well as, the relief of pain associated with tension andmigraine headaches and menstrual cramps. Chronic conditions includearthritic diseases such as rheumatoid arthritis and osteoarthritis.Although rheumatoid arthritis is largely an autoimmune disease andosteoarthritis is caused by the degradation of cartilage in joints,reducing the inflammation associated with each provides a significantincrease in the quality of life for those suffering from these diseases(Wienberg (2001) Immunol. Res. 22:319-41; Wollhiem (2000) Curr. Opin.Rheum. 13:193-201). As inflammation is a component of rheumatic diseasesin general, the use of COX inhibitors has been expanded to includediseases such as systemic lupus erythromatosus (SLE) (Goebel et al.(1999) Chem. Res. Tox. 12:488-500; Patrono et al. (1985) J. Clin.Invest. 76:1011-1018) and rheumatic skin conditions such as scleroderma.COX inhibitors are also used for the relief of inflammatory skinconditions that are not of rheumatic origin, such as psoriasis, in whichreducing the inflammation resulting from the over production ofprostaglandins could provide a direct benefit (Fogh et al. (1993) ActaDerm. Venereol (Oslo) 73:191-3).

In addition to their use as anti-inflammatory agents, another potentialrole for COX inhibitors is the treatment of cancer. Over-expression ofCOX-2 has been demonstrated in various human malignancies and inhibitorsof COX-2 have been shown to be efficacious in the treatment of animalswith skin, breast and bladder tumors. While the mechanism of action isnot completely understood, the over-expression of COX-2 has been shownto inhibit apoptosis and increase the invasiveness of tumorgenic celltypes (Dempke et al. (2001) J. Can. Res. Clin. Oncol. 127:411-17; Mooreand Simmons (2000) Current Med. Chem. 7:1131-44). It is possible thatenhanced production of prostaglandins, resulting from theover-expression of COX-2, promotes cellular proliferation andconsequently increases angiogenesis. (Moore (1985) in Prostanoids:Pharmacological, Physiological and Clinical Relevance, CambridgeUniversity Press, N.Y., pp. 229-30; Fenton et al. (2001) Am. J. Clin.Oncol. 24:453-57).

There have been a number of clinical studies evaluating COX-2 inhibitorsfor potential use in the prevention and treatment of different types ofcancer. In 1999, 130,000 new cases of colorectal cancer were diagnosedin the United States. Aspirin, a non-specific NSAID, has been found toreduce the incidence of colorectal cancer by 40-50% (Giovannucci et al.(1995) N. Engl. J. Med. 333:609-614) and mortality by 50% (Smalley etal. (1999) Arch. Intern. Med. 159:161-166). In 1999, the FDA approvedthe COX-2 inhibitor celecoxib for use in FAP (Familial AdemonatousPolyposis) to reduce colorectal cancer mortality. It is believed thatother cancers with evidence of COX-2 involvement may be successfullyprevented and/or treated with COX-2 inhibitors including, but notlimited to, esophageal cancer, head and neck cancer, breast cancer,bladder cancer, cervical cancer, prostate cancer, hepatocellularcarcinoma and non-small cell lung cancer (Jaeckel et al. (2001) Arch.Otolamygol. 127:1253-59; Kirschenbaum et al. (2001) Urology 58:127-31;Dannhardt and Kiefer (2001) Eur. J. Med. Chem. 36:109-26). COX-2inhibitors may also prove successful in preventing colon cancer inhigh-risk patients. There is also evidence that COX-2 inhibitors canprevent or even reverse several types of life-threatening cancers. Todate, as many as fifty studies show that COX-2 inhibitors can preventpre-malignant and malignant tumors in animals, and possibly preventbladder, esophageal and skin cancers as well. COX-2 inhibition couldprove to be one of the most important preventive medical accomplishmentsof the century.

Recent scientific progress has identified correlations between COX-2expression, general inflammation and the pathogenesis of Alzheimer'sDisease (AD) (Ho et al. (2001) Arch. Neurol. 58:487-92). In animalmodels, transgenic mice that over-express the COX-2 enzyme have neuronsthat are more susceptible to damage. The National Institute on Aging(NIA) is launching a clinical trial to determine whether NSAIDs can slowthe progression of Alzheimer's disease. Naproxen (a non-selective NSAID)and rofecoxib (Vioxx, a COX-2 specific selective NSAID) will beevaluated. Previous evidence has indicated that inflammation contributesto Alzheimer's disease. According to the Alzheimer's Association and theNIA, about 4 million people suffer from AD in the United States and thisis expected to increase to 14 million by mid-century.

The COX enzyme (also known as prostaglandin H₂ synthase) catalyzes twoseparate reactions. In the first reaction, AA is metabolized to form theunstable prostaglandin G₂ (PGG₂), a cyclooxygenase reaction. In thesecond reaction, PGG₂ is converted to the endoperoxide PGH₂, aperoxidase reaction. The short-lived PGH₂ non-enzymatically degrades toPGE₂. The compounds described herein are the result of a discoverystrategy that combined an assay focused on the inhibition of COX-1 andCOX-2 peroxidase activity with a chemical dereplication process toidentify novel inhibitors of the COX enzymes.

The term gene expression is often used to describe the broad result ofmRNA production and protein synthesis. In fact, changes in actual geneexpression may never result in observable changes on the protein level.The corollary, that changes in protein level do not always result fromchanges in gene expression, can also be true. There are six possiblepoints of regulation in the pathway leading from genomic DNA to afunctional protein: (1) transcriptional regulation by nuclear factorsand other signals leading to production of pre-mRNA; (2) pre-mRNAprocessing regulation involving exon splicing, the additions of a 5′ capstructure and 3′ poly-adenylation sequence and transport of the maturemRNA from the nucleus into the cytoplasm; (3) mRNA transport regulationcontrolling localization of the mRNA to a specific cytoplasmic site fortranslation into protein; (4) mRNA degradation regulation controllingthe size of the mRNA pool either prior to any protein translation or asa means of ending translation from that specific mRNA; (5) translationalregulation of the specific rate of protein translation initiation and(6) post-translation processing regulation involving modifications suchas glycosylation and proteolytic cleavage. In the context of genomicsresearch it is important to use techniques that measure gene expressionlevels closer to the initial steps (e.g. mRNA levels) rather than latersteps (e.g. protein levels) in this pathway.

Recent reports have addressed the possible involvement of flavonoids,isolated from the medicinal herb Scutellaria baicalensis, in alterationsin cox-2 gene expression (Wakabayashi and Yasui (2000) Eur. J.Pharmacol. 406:477-481; Chen et al. (2001) Biochem. Pharmacol.61:1417-1427; Chi et al. (2001) 61:1195-1203 and Raso et al. (2001) LifeSci. 68:921-931). Each of above cited studies on cox-2 gene expressionused a Western Blot technique to evaluate putative alterations in geneexpression without validation on the molecular level. Since this methodonly measures protein levels and not the specific transcription product,mRNA, it is possible that other mechanisms are involved leading to theobserved increase in protein expression. For example, LPS has beenreported to modulate mRNA half-lives via instability sequences found inthe 3′ untranslated region (3′UTR) of mRNAs (Watkins et al. (1999) LifeSci. 65:449-481), which could account for increased protein expressionwithout alternations in the rate of gene transcription. Consequently,this leaves open the question of whether or not these treatmentconditions resulted in a meaningful change in gene expression.

Techniques, such as RT-qPCR and DNA microarray analysis, rely on mRNAlevels for analysis and can be used to evaluate levels of geneexpression under different conditions, i.e. in the presence or absenceof a pharmaceutical agent. There are no known reports using techniquesthat specifically measure the amount of mRNA, directly or indirectly, inthe literature when Free-B-ring flavonoids or flavans are used as thetherapeutic agents.

Flavonoids are a widely distributed group of natural products. Theintake of flavonoids has been demonstrated to be inversely related tothe risk of incident dementia. The mechanism of action, while not known,has been speculated as being due to the anti-oxidative effects offlavonoids (Commenges et al. (2000) Eur. J. Epidemiol. 16:357-363).Polyphenol flavones induce programmed cell death, differentiation andgrowth inhibition in transformed colonocytes by acting at the mRNA levelon genes including cox-2, Nuclear Factor kappa B (NFκB) and bcl-X(L)(Wenzel et al. (2000) Cancer Res. 60:3823-3831). It has been reportedthat the number of hydroxyl groups on the B ring is important in thesuppression of cox-2 transcriptional activity (Mutoh et al. (2000) Jnp.J. Cancer Res. 91:686-691).

Free-B-ring flavones and flavonols are a specific class of flavonoids,which have no substituent groups on the aromatic B ring (referred toherein as Free-B-ring flavonoids), as illustrated by the followinggeneral structure:

wherein

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

Free-B-ring flavonoids are relatively rare. Out of 9,396 flavonoidssynthesized or isolated from natural sources, only 231 Free-B-ringflavonoids are known (The Combined Chemical Dictionary, Chapman &Hall/CRC, Version 5: Jun. 1, 2001). Free-B-ring flavonoids have beenreported to have diverse biological activity. For example, galangin(3,5,7-trihydroxyflavone) acts as an anti-oxidant and free radicalscavenger and is believed to be a promising candidate foranti-genotoxicity and cancer chemoprevention. (Heo et al. (2001) Mutat.Res. 488:135-150). It is an inhibitor of tyrosinase monophenolase (Kuboet al. (2000) Bioorg. Med. Chem. 8:1749-1755), an inhibitor of rabbitheart carbonyl reductase (Imamura et al. (2000) J. Biochem.127:653-658), has antimicrobial activity (Afolayan and Meyer (1997)Ethnopharmacol. 57:177-181) and antiviral activity (Meyer et al. (1997)J. Ethnopharmacol. 56:165-169). Baicalein and two other Free-B-ringflavonoids, have antiproliferative activity against human breast cancercells. (So et al. (1997) Cancer Lett. 112:127-133).

Typically, flavonoids have been tested for activity randomly based upontheir availability. Occasionally, the requirement of substitution on theB-ring has been emphasized for specific biological activity, such as theB-ring substitution required for high affinity binding to p-glycoprotein(Boumendjel et al. (2001) Bioorg. Med. Chem. Lett. 11:75-77);cardiotonic effect (Itoigawa et al. (1999) J. Ethnopharmacol.65:267-272), protective effect on endothelial cells against linoleicacid hydroperoxide-induced toxicity (Kaneko and Baba (1999) Biosci.Biotechnol. Biochem. 63:323-328), COX-1 inhibitory activity (Wang (2000)Phytomedicine 7:15-19) and prostaglandin endoperoxide synthase activity(Kalkbrenner et al. (1992) Pharmacology 44:1-12). Only a fewpublications have mentioned the significance of the unsubstituted B-ringof the Free-B-ring flavonoids. One example is the use of 2-phenylflavones, which inhibit NADPH quinone acceptor oxidoreductase, aspotential anticoagulants (Chen et al. (2001) Biochem. Pharmacol.61:1417-1427).

The reported mechanism of action with respect to the anti-inflammatoryactivity of various Free-B-ring flavonoids has been controversial. Theanti-inflammatory activity of the Free-B-ring flavonoids, chrysin (Lianget al. (2001) FEBS Lett. 496:12-18), wogonin (Chi et al. (2001) Biochem.Pharmacol. 61:1195-1203) and halangin (Raso et al. (2001) Life Sci.68:921-931) has been associated with the suppression of induciblecyclooxygenase and nitric oxide synthase via activation ofperoxisome-proliferator activated receptor gamma (PPARγ) and influenceon degranulation and AA release (Tordera et al. (1994) Z. Naturforsch[C] 49:235-240). It has been reported that oroxylin, baicalein andwogonin inhibit 12-lipoxygenase activity without affectingcyclooxygenases (You et al. (1999) Arch. Pharm. Res. 22:18-24). Morerecently, the anti-inflammatory activity of wogonin, baicalin andbaicalein has been reported as occurring through inhibition of induciblenitric oxide synthase and cox-2 enzyme production induced by nitricoxide inhibitors and lipopolysaccharides (Chen et al. (2001) Biochem.Pharmacol. 61:1417-1427). It has also been reported that oroxylin actsvia suppression of NFκB activation (Chen et al. (2001) Biochem.Pharmacol. 61:1417-1427). Finally, wogonin reportedly inhibits induciblePGE₂ production in macrophages (Wakabayashi and Yasui (2000) Eur. J.Pharmacol. 406:477-481).

Inhibition of the phosphorylation of mitogen-activated protein kinase(MAPK) and inhibition of Ca²⁺ ionophore A23187 induced PGE₂ release bybaicalein has been reported as the mechanism of anti-inflammatoryactivity of Scutellariae radix (Nakahata et al. (1999) Nippon YakurigakuZasshi 114, Supp. 11:215P-219P; Nakahata et al. (1998) Am. J. Chin. Med.26:311-323). Baicalin from Scutellaria baicalensis reportedly inhibitssuperantigenic staphylococcal exotoxins stimulated T-cell proliferationand production of IL-1β, IL-6, tumor necrosis factor-α (TNF-α), andinterferon-γ (IFN-γ (Krakauer et al. (2001) FEBS Lett. 500:52-55). Thus,the anti-inflammatory activity of baicalin has been associated withinhibiting the pro-inflammatory cytokines mediated signaling pathwaysactivated by superantigens. However, it has also been proposed that theanti-inflammatory activity of baicalin is due to the binding of avariety of chemokines, which limit their biological activity (Li et al.(2000) Immunopharmacol. 49:295-306). Recently, the effects of baicalinon adhesion molecule expression induced by thrombin and thrombinreceptor agonist peptide (Kimura et al. (2001) Planta Med. 67:331-334),as well as, the inhibition of MAPK cascade (Nakahata et al. (1999)Nippon Yakurigaku Zasshi 114, Supp 11:215P-219P; Nakahata et al. (1998)Am. J. Chin Med. 26:311-323) have been reported.

The Chinese medicinal plant Scutellaria baicalensis contains significantamounts of Free-B-ring flavonoids, including baicalein, baicalin,wogonin and baicalenoside. Traditionally, this plant has been used totreat a number of conditions including clearing away heat, purging fire,dampness-warm and summer fever syndromes; polydipsia resulting from highfever; carbuncle, sores and other pyogenic skin infections; upperrespiratory infections such as acute tonsillitis, laryngopharyngitis andscarlet fever; viral hepatitis; nephritis; pelvitis; dysentery;hematemesis and epistaxis. This plant has also traditionally been usedto prevent miscarriage (see Encyclopedia of Chinese TraditionalMedicine, ShangHai Science and Technology Press, ShangHai, China, 1998).Clinically, Scutellaria is now used to treat conditions such aspediatric pneumonia, pediatric bacterial diarrhea, viral hepatitis,acute gallbladder inflammation, hypertension, topical acute inflammationresulting from cuts and surgery, bronchial asthma and upper respiratoryinfections (Encyclopedia of Chinese Traditional Medicine, ShangHaiScience and Technology Press, ShangHai, China, 1998). Thepharmacological efficacy of Scutellaria roots for treating bronchialasthma is reportedly related to the presence of Free-B-ring flavonoidsand their suppression of eotaxin associated recruitment of eosinophils(Nakajima et al. (2001) Planta Med. 67(2):132-135).

To date, a number of naturally occurring Free-B-ring flavonoids havebeen commercialized for varying uses. For example, liposome formulationsof Scutellaria extracts have been utilized for skin care (U.S. Pat. Nos.5,643,598; 5,443,983). Baicalin has been used for preventing cancer dueto its inhibitory effects on oncogenes (U.S. Pat. No. 6,290,995).Baicalin and other compounds have been used as antiviral, antibacterialand immunomodulating agents (U.S. Pat. No. 6,083,921) and as naturalanti-oxidants (Poland Pub. No. 9,849,256). Chrysin has been used for itsanxiety reducing properties (U.S. Pat. No. 5,756,538). Anti-inflammatoryflavonoids are used for the control and treatment of anorectal andcolonic diseases (U.S. Pat. No. 5,858,371) and inhibition oflipoxygenase (U.S. Pat. No. 6,217,875). These compounds are alsoformulated with glucosamine collagen and other ingredients for repairand maintenance of connective tissue (U.S. Pat. No. 6,333,304).Flavonoid esters constitute the active ingredients for cosmeticcompositions (U.S. Pat. No. 6,235,294). U.S. application Ser. No.10/091,362, filed Mar. 1, 2002, entitled “Identification of Free-B-ringFlavonoids as Potent COX-2 Inhibitors,” discloses a method forinhibiting the cyclooxygenase enzyme COX-2 by administering acomposition comprising a Free-B-ring flavonoid or a compositioncontaining a mixture of Free-B-ring flavonoids to a host in needthereof. This is the first report of a link between Free-B-ringflavonoids and COX-2 inhibitory activity. This application isspecifically incorporated herein by reference in its entirety.

Japanese Pat. No. 63027435, describes the extraction, and enrichment ofbaicalein and Japanese Pat. No. 61050921 describes the purification ofbaicalin.

Flavans include compounds illustrated by the following generalstructure:

wherein

R₁, R₂, R₃, R₄ and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂,—NR₃ ⁺X—, esters of the mentioned substitution groups, including, butnot limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoylesters, trihydroxybenzoyl esters and caffeoyl esters, and their chemicalderivatives thereof; a carbon, oxygen, nitrogen or sulfur glycoside of asingle or a combination of multiple sugars including, but not limitedto, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof; dimer, trimer and other polymerizedflavans;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, and carbonate, etc.

Catechin is a flavan, found primarily in Acacia, having the followingstructure

Catechin works both alone and in conjunction with other flavonoids foundin tea, and has both antiviral and antioxidant activity. Catechin hasbeen shown to be effective in the treatment of viral hepatitis. It alsoappears to prevent oxidative damage to the heart, kidney, lungs andspleen and has been shown to inhibit the growth of stomach cancer cells.

Catechin and its isomer epicatechin inhibit prostaglandin endoperoxidesynthase with an IC₅₀ value of 40 μM. (Kalkbrenner et al. (1992)Pharmacol. 44:1-12). Five flavan-3-ol derivatives, including(+)-catechin and gallocatechin, isolated from the four plant species,Atuna racemosa, Syzygium carynocarpum, Syzygium malaccense and Vantaneaperuviana, exhibit equal to or weaker inhibitory activity against COX-2,relative to COX-1, with IC₅₀ values ranging from 3.3 μM to 138 μM(Noreen et al. (1998) Planta Med. 64:520-524). (+)-Catechin, isolatedfrom the bark of Ceiba pentandra, inhibits COX-1 with an IC₅₀ value of80 μM (Noreen et al. (1998) J. Nat. Prod. 61:8-12). Commerciallyavailable pure (+)-catechin inhibits COX-1 with an IC₅₀ value of around183 to 279 μM, depending upon the experimental conditions, with noselectivity for COX-2 (Noreen et al. (1998) J. Nat. Prod. 61:1-7).

Green tea catechin, when supplemented into the diets of Sprague dawleymale rats, lowered the activity level of platelet PLA₂ and significantlyreduced platelet cyclooxygenase levels (Yang et al. (1999) J. Nutr. Sci.Vitaminol. 45:337-346). Catechin and epicatechin reportedly weaklysuppress cox-2 gene transcription in human colon cancer DLD-1 cells(IC₅₀=415.3 μM) (Mutoh et al. (2000) Jpn. J. Cancer Res. 91:686-691).The neuroprotective ability of (+)-catechin from red wine results fromthe antioxidant properties of catechin, rather than inhibitory effectson intracellular enzymes, such as cyclooxygenase, lipoxygenase or nitricoxide synthase (Bastianetto et al. (2000) Br. J. Pharmacol.131:711-720). Catechin derivatives purified from green tea and blacktea, such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC),epicatechin-3-gallate (ECG) and theaflavins showed inhibition ofcyclooxygenase- and lipoxygenase-dependent metabolism of AA in humancolon mucosa and colon tumor tissues (Hong et al. (2001) Biochem.Pharmacol. 62:1175-1183) and induced cox-2 gene expression and PGE₂production (Park et al. (2001) Biochem. Biophys. Res. Commun.286:721-725). Epiafzelechin isolated from the aerial parts of Celastrusorbiculatus exhibited dose-dependent inhibition of COX-1 activity withan IC₅₀ value of 15 μM and also demonstrated anti-inflammatory activityagainst carrageenin-induced mouse paw edema following oraladministration at a dosage of 100 mg/kg (Min et al. (1999) Planta Med.65:460-462).

Catechin and its derivatives from various plant sources, especially fromgreen tea leaves, have been used in the treatment of HPV infectedCondyloma acuminata (Cheng, U.S. Pat. No. 5,795,911) and in thetreatment of hyperplasia caused by papilloma virus (Cheng, U.S. Pat.Nos. 5,968,973 and 6,197,808). Catechin and its derivatives have alsobeen used topically to inhibit angiogenesis in mammalian tissue, inconditions such as skin cancer, psoriasis, spider veins or under eyecircles (Anderson, U.S. Pat. No. 6,248,341), against UVB-inducedtumorigenesis in mice (Agarwal et al. (1993) Photochem. Photobiol.58:695-700), for inhibiting nitric oxide synthase at the level of geneexpression and enzyme activity (Chan, U.S. Pat. No. 5,922,756), and ashair-growing agents (Takahashi, U.S. Pat. No. 6,126,940). Catechin-basedcompositions have also been formulated with other extracts and vitaminsfor treatment of acne (Murad, U.S. Pat. No. 5,962,517), hardening thetissue of digestive organs (Shi, U.S. Pat. No. 5,470,589) and forinhibiting 5 alpha-reductase activity in treating androgenic disorderrelated diseases and cancers (Liao, U.S. Pat. No. 5,605,929). Green teaextract has been formulated with seven other plant extracts for reducinginflammation by inhibiting the COX-2 enzyme, without identification ofany of the specific active components (Mewmark, U.S. Pat. No.6,264,995).

Acacia is a genus of leguminous trees and shrubs. The genus Acaciaincludes more than 1,000 species belonging to the family of Leguminosaeand the subfamily of Mimosoideae. Acacias are distributed worldwide inplaces such as tropical and subtropical areas of Central and SouthAmerica, Africa, parts of Asia, as well as, Australia, which has thelargest number of endemic species. Acacias are present primarily in dryand arid regions where the forests are often in the nature of openthorny shrubs. The genus Acacia is divided into 3 subgenera based mainlyon leaf morphology—Acacia, Aculiferum and Heterophyllum. Based on thenature of the leaves of mature trees, however, the genus Acacia can bedivided into two “popular” groups—the typical bipinnate-leaved speciesand the phyllodenous species. A phyllode is a modified petiole expandedinto a leaf-like structure with no leaflets, an adaptation to xerophyticconditions. The typical bipinnate-leaved species are found primarilythroughout the tropics, whereas the phyllodenous species occur mainly inAustralia. More than 40 species of Acacia have been reported in India.Gamble in his book entitled Flora of Madras Presidency listed 23 nativespecies for southern India, 15 of which are found in Tamil Nadu. Sincethat time, however, many new Acacia species have been introduced toIndia and approximately 40 species are now found in Tamil Nadu itself.The indigenous species are primarily thorny trees or shrubs and a feware thorny stragglers, such as A. caesia, A. pennata and A. sinuata.Many species have been introduced from Africa and Australia, includingA. mearnsii, A. picnantha and A. dealbata, which have bipinnate leavesand A. auriculiformis, A. holoserecia and A. mangium, which arephyllodenous species.

Acacias are very important economically, providing a source of tannins,gums, timber, fuel and fodder. Tannins, which are isolated primarilyfrom the bark, are used extensively for tanning hides and skins. SomeAcacia barks are also used for flavoring local spirits. Some indigenousspecies like A. sinuata also yield saponins, which are any of variousplant glucosides that form soapy lathers when mixed and agitated withwater. Saponins are used in detergents, foaming agents and emulsifiers.The flowers of some Acacia species are fragrant and used to makeperfume. For example, cassie perfume is obtained from A. ferrugenea. Theheartwood of many Acacias is used for making agricultural implements andalso provides a source of firewood. Acacia gums find extensive use inmedicine and confectionary and as sizing and finishing materials in thetextile industry. Lac insects can be grown on several species, includingA. nilotica and A. catechu. Some species have been used for forestationof wastelands, including A. nilotica, which can withstand some waterinundation and a few such areas have become bird sanctuaries.

To date, approximately 330 compounds have been isolated from variousAcacia species. Flavonoids, a type of water-soluble plant pigments, arethe major class of compounds isolated from Acacias. Approximately 180different flavonoids have been identified, 111 of which are flavans.Terpenoids are second largest class of compounds isolated from speciesof the Acacia genus, with 48 compounds having been identified. Otherclasses of compounds isolated from Acacia include, alkaloids (28), aminoacids/peptides (20), tannins (16), carbohydrates (15), oxygenheterocycles (15) and aliphatic compounds (10). (Buckingham, in TheCombined Chemical Dictionary, Chapman & Hall CRC, version 5:2, December2001).

Phenolic compounds, particularly flavans are found in moderate to highconcentrations in all Acacia species (Abdulrazak et al. (2000) J. Anim.Sci. 13:935-940). Historically, most of the plants and extracts of theAcacia genus have been utilized as astringents to treat gastrointestinaldisorders, diarrhea, indigestion and to stop bleeding (Vautrin (1996)Universite Bourgogne (France) European abstract 58-01C:177; Saleem etal. (1998) Hamdard Midicus. 41:63-67). The bark and pods of A. arabicaWilld. contain large quantities of tannins and have been utilized asastringents and expectorants (Nadkarni (1996) India Materia Medica,Bombay Popular Prakashan, pp. 9-17). Diarylpropanol derivatives,isolated from stem bark of A. tortilis from Somalia, have been reportedto have smooth muscle relaxing effects (Hagos et al. (1987) Planta Med.53:27-31, 1987). It has also been reported that terpenoid saponinsisolated from A. victoriae have an inhibitory effect ondimethylbenz(a)anthracene-induced murine skin carcinogenesis (Hanauseket al. (2000) Proc. Am. Assoc. Can. Res. Annu. Mtg. 41:663) and induceapoptosis (Haridas et al. (2000) Proc. Am. Assoc. for Can. Res. Annu.Mtg. 41:600). Plant extracts from A. nilotica have been reported to havespasmogenic, vasoconstrictor and anti-hypertensive activity (Amos et al.(1999) Phytotherapy Research 13:683-685; Gilani et al. (1999)Phytotherapy Research 13:665-669), and antiplatelet aggregatory activity(Shah et al. (1997) Gen. Pharmacol. 29:251-255). Anti-inflammatoryactivity has been reported for A. nilotica. It was speculated thatflavonoids, polysaccharides and organic acids were potential activecomponents (Dafallah and Al-Mustafa (1996) Am. J. Chin. Med.24:263-269). To date, the only reported 5-lipoxygenase inhibitorisolated from Acacia is a monoterpenoidal carboxamide (Seikine et al.(1997) Chem. Pharm. Bull. (Tokyo) 45:148-11).

Acacia gums have been formulated with other plant ingredients and usedfor ulcer prevention without identification of any of the activecomponents (Fuisz, U.S. Pat. No. 5,651,987). Acacia gums have also beenformulated with other plant ingredients and used to improve drugdissolution (Blank, U.S. Pat. No. 4,946,684), by lowering the viscosityof nutritional compositions (Chancellor, U.S. Pat. No. 5,545,411).

The extract from the bark of Acacia was patented in Japan for externaluse as a whitening agent (Abe, JP10025238), as a glucosyl transferaseinhibitor for dental applications (Abe, JP07242555), as a proteinsynthesis inhibitor (Fukai, JP 07165598), as an active oxygen-scavengingagent for external skin preparations (Honda, JP 07017847, Bindra U.S.Pat. No. 61/266,950), and as a hyaluronidase inhibitor for oralconsumption to prevent inflammation, pollinosis and cough (Ogura, JP07010768).

Review of the literature has revealed no human clinical applicationsusing mixtures of Free-B-ring flavonoids and flavans for relief of painor measuring biochemical clinical outcomes for osteoarthritis treatment.This report appears to be the first randomized, double blind, placebocontrolled study of the safety and efficacy of these compounds inhumans.

SUMMARY OF THE INVENTION

The present invention includes a novel composition of matter comprisedof a mixture of Free-B-ring flavonoids and flavans. This novelcomposition of matter is referred to herein as Univestin™. The ratio ofFree-B-ring flavonoids to flavans in the composition of matter can beadjusted based on the indications and the specific requirements withrespect to prevention and treatment of a specific disease or condition.Generally, the ratio of Free-B-ring flavonoids to flavans can be in therange of 99:1 Free-B-ring flavonoids:flavans to 1:99 of Free-B-ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodimentthe Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

The present invention further includes methods that are effective insimultaneously inhibiting both the COX-2 and 5-LO enzymes. The methodfor the simultaneous dual inhibition of the COX-2 and 5-LO pathways iscomprised of administering a composition comprising a mixture ofFree-B-ring flavonoids and flavans synthesized and/or isolated from asingle plant or multiple plants to a host in need thereof. The efficacyof this method was demonstrated with purified enzymes, in different celllines, multiple animal models and eventually in a human clinical study.The ratio of Free-B-ring flavonoids to flavans in the composition can bein the range of 99:1 Free-B-ring flavonoids:flavans to 1:99 ofFree-B-ring flavonoids:flavans. In specific embodiments of the presentinvention, the ratio of Free-B-ring flavonoids to flavans is selectedfrom the group consisting of approximately 90:10, 80:20, 70:30, 60:40,50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodiment,the Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

The present invention further includes methods for the prevention andtreatment of COX-2 and 5-LO mediated diseases and conditions, includingbut not limited to menstrual cramps, arteriosclerosis, heart attack,obesity, diabetes, syndrome X, Alzheimer's disease, respiratory allergicreaction, chronic venous insufficiency, hemorrhoids, Systemic LupusErythromatosis, psoriasis, chronic tension headache, migraine headaches,inflammatory bowl disease; topical infections caused by virus, bacteriaand fungus, sunburn, thermal burns, contact dermatitis, melanoma andcarcinoma. The method for preventing and treating COX-2 and 5-LOmediated diseases and conditions is comprised of administering to a hostin need thereof an effective amount of a composition comprising amixture of Free-B-ring flavonoids and flavans synthesized and/orisolated from a single plant or multiple plants together with apharmaceutically acceptable carrier. The ratio of Free-B-ring flavonoidsto flavans can be in the range of 99:1 Free-B-ring flavonoids:flavans to1:99 of Free-B-ring flavonoids:flavans. In specific embodiments of thepresent invention, the ratio of Free-B-ring flavonoids to flavans isselected from the group consisting of approximately 90:10, 80:20, 70:30,60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodimentof the invention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodiment,the Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

In another embodiment, the present invention includes a method fortreating general joint pain and stiffness, improving mobility andphysical function and preventing and treating pathological conditions ofosteoarthritis and rheumatoid arthritis. The method for preventing andtreating joint pain and stiffness, improving mobility and physicalfunction and preventing and treating pathological conditions ofosteoarthritis, and rheumatoid arthritis comprises administering to ahost in need thereof an effective amount of a composition comprising amixture of Free-B-ring flavonoids and flavans synthesized and/orisolated from a single plant or multiple plants together with apharmaceutically acceptable carrier. The ratio of Free-B-ring flavonoidsto flavans can be in the range of 99:1 to 1:99 Free-B-ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodiment,the Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

The present invention includes methods for weight loss and blood sugarcontrol due to increased physical activity resulting from improvingmobility, flexibility and physical function said method comprisingadministering to a host in need thereof an effective amount of acomposition comprising a mixture of Free-B-ring flavonoids and flavanssynthesized and/or isolated from a single plant or multiple plants and apharmaceutically acceptable carrier. The ratio of Free-B-ring flavonoidsto flavans can be in the range of 99:1 Free-B-ring flavonoids:flavans to1:99 of Free-B-ring flavonoids:flavans. In specific embodiments of thepresent invention, the ratio of Free-B-ring flavonoids to flavans isselected from the group consisting of approximately 90:10, 80:20, 70:30,60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodimentof the invention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodimentthe Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

The present invention also includes a method for modulating theproduction of mRNA implicated in pain pathways said method comprisingadministering to a host in need thereof an effective amount of acomposition comprising a mixture of Free-B-ring flavonoids and flavanssynthesized and/or isolated from a single plant or multiple plants and apharmaceutically acceptable carrier. While not limited by theory,Applicant believes that the ability to modulate the production of mRNAis accomplished via a decrease, by the active ingredients in theFree-B-ring/flavan composition, in the production of mRNA by the cox-2gene, but not the cox-1 gene. The ratio of Free-B-ring flavonoids toflavans in the composition can be in the range of 99:1 to 1:99Free-B-ring flavonoids:flavans. In specific embodiments of the presentinvention, the ratio of Free-B-ring flavonoids to flavans is selectedfrom the group consisting of approximately 90:10, 80:20, 70:30, 60:40,50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodimentthe Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

The Free-B-ring flavonoids, also referred to herein as Free-B-ringflavones and flavonols, that can be used in accordance with thefollowing invention include compounds illustrated by the followinggeneral structure:

wherein

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

The flavans that can be used in accordance with the following inventioninclude compounds illustrated by the following general structure:

wherein

R₁, R₂, R₃, R₄ and R₅ are independently selected from the groupconsisting of H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂,—NR₃ ⁺X⁻, esters of the mentioned substitution groups, including, butnot limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoylesters, trihydroxybenzoyl esters and caffeoyl esters and their chemicalderivatives thereof; carbon, oxygen, nitrogen or sulfur glycoside of asingle or a combination of multiple sugars including, but not limitedto, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof; dimer, trimer and other polymerizedflavans;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

The Free-B-ring flavonoids of this invention may be obtained bysynthetic methods or extracted from the families of plants including,but not limited to Annonaceae, Asteraceae, Bignoniaceae, Combretaceae,Compositae, Euphorbiaceae, Labiatae, Lauranceae, Leguminosae, Moraceae,Pinaceae, Pteridaceae, Sinopteridaceae, Ulmaceae and Zingiberaceae. TheFree-B-ring flavonoids can be extracted, concentrated, and purified fromthe genera of high plants, including but not limited to Desmos,Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium,Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria,Molsa, Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne,Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus,Ficus, Pityrogramma, Notholaena, Pinus, Ulmus and Alpinia.

As noted above the flavans of this invention may be obtained from aplant or plants selected from the genus of Acacia. In a preferredembodiment, the plant is selected from the group consisting of Acaciacatechu, A. concinna, A. farnesiana, A. Senegal, A. speciosa, A.arabica, A. caesia, A. pennata, A. sinuata. A. mearnsii, A. picnantha,A. dealbata, A. auriculiformis, A. holoserecia and A. mangium.

The present invention includes an evaluation of different compositionsof Free-B-ring flavonoids and flavans using enzymatic and in vivo modelsto optimize the formulation and obtain the best potency. The efficacyand safety of this formulation is also demonstrated in human clinicalstudies. The present invention provides a commercially viable processfor the isolation, purification and combination of Acacia flavans withFree-B-ring flavonoids to yield composition of matter having desirablephysiological activity. The compositions of this invention can beadministered by any method known to one of ordinary skill in the art.The modes of administration include, but are not limited to, enteral(oral) administration, parenteral (intravenous, subcutaneous, andintramuscular) administration and topical application. The method oftreatment according to this invention comprises administering internallyor topically to a patient in need thereof a therapeutically effectiveamount of a mixture of Free-B-ring flavonoids and flavans synthesizedand/or isolated from a single plant or multiple plants.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts graphically the inhibition of COX-1 and COX-2 by HTPfractions from Acacia catechu. The extracts were prepared andfractionated as described in Examples 1 and 3. The extracts wereexamined for their inhibition of the peroxidase activity of recombinantovine COX-1 (▪) or ovine COX-2 (♦) as described in Example 2. The datais presented as percent of untreated control.

FIG. 2 depicts graphically the inhibition of COX-1 and COX-2 by HTPfractions from Scutellaria baicalensis. The extracts were prepared andfractionated as described in Examples 1 and 3. The extracts wereexamined for their inhibition of the peroxidase activity of recombinantovine COX-1 (▪) or ovine COX-2 (♦) as described in Example 2. The datais presented as percent of untreated control.

FIG. 3 depicts a HPLC chromatogram of a standardized extract isolatedfrom the roots of Scutellaria baicalensis (lot # RM052302-01) having aFree-B-ring flavonoid content of 82.2%. Ten structures were elucidatedusing HPLC/PDA/MS as baicalin, wogonin-7-glucuronide, oroxylin A7-glucuronide, baicalein, wogonin, chrysin-7-glucuronide,norwogonin-7-glucuronide, scutellarin, chrysin and oroxylin A.

FIG. 4 depicts graphically a profile of the inhibition of COX-1 andCOX-2 by the baicalein, which was isolated and purified from Scutellariabaicalensis. The compound was examined for its inhibition of theperoxidase activity of recombinant ovine COX-1 (♦) and ovine COX-2 (▪).The data is presented as percent inhibition of assays without inhibitorvs. inhibitor concentration (μg/mL). The IC₅₀ for COX-1 was calculatedas 0.18 μg/mL/unit of enzyme while the IC₅₀ for COX-2 was calculated as0.28 μg/mL/unit.

FIG. 5 depicts graphically a profile of the inhibition of COX-1 andCOX-2 by the baicalin, which was isolated and purified from Scutellariabaicalensis. The compound was examined for its inhibition of theperoxidase activity of recombinant ovine COX-1 (♦) and ovine COX-2 (▪).The data is presented as percent inhibition of assays without inhibitorvs. inhibitor concentration (μg/mL). The IC₅₀ for COX-1 was determinedto be 0.44 μg/mL/unit of enzyme while that of COX-2 was determined to be0.28 μg/mL/unit.

FIG. 6 depicts graphically a profile of the inhibition of COX-1 andCOX-2 by a standardized Free-B-ring flavonoid extract (83% baicalinbased on HPLC) isolated from Scutellaria baicalensis. The extract wasexamined for its inhibition of the peroxidase activity of recombinantovine COX-1 (♦) and ovine COX-2 (▪). The data is presented as percentinhibition of assays without inhibitor vs. inhibitor concentration(μg/mL). The IC₅₀ for COX-1 was calculated as 0.24 μg/mL/unit of enzymewhile the IC₅₀ for COX-2 was calculated as 0.48 μg/mL/unit.

FIG. 7 depicts graphically a profile of the inhibition of COX-1 andCOX-2 by catechin, which was isolated and purified from Acacia catechu.The compound was examined for its inhibition of the peroxidase activityof recombinant ovine COX-1 (♦) and ovine COX-2 (▪). The data ispresented as percent inhibition of assays without inhibitor vs.inhibitor concentration (μg/mL). The IC₅₀ for COX-1 was determined to be0.11 μg/mL/unit of enzyme while the IC₅₀ for COX-2 was determined as0.42 μg/mL/unit.

FIG. 8 depicts graphically a profile of the inhibition of COX-1 andCOX-2 by a standardized flavan extract containing 50% total catechinsisolated from Acacia catechu. The extract was examined for itsinhibition of the peroxidase activity of recombinant ovine COX-1 (♦) andovine COX-2 (▪). The data is presented as percent inhibition of assayswithout inhibitor vs. inhibitor concentration (μg/mL). The IC₅₀ forCOX-1 was calculated as 0.17 μg/mL/unit of enzyme while the IC₅₀ forCOX-2 was determined to be 0.41 μg/mL/unit.

FIG. 9 depicts the HPLC chromatogram of the flavans extracted fromAcacia catechu with 80% MeOH in water.

FIG. 10 depicts graphically a profile of the inhibition of 5-LO by thepurified flavan catechin from Acacia catechu. The compound was examinedfor its inhibition of recombinant potato 5-lipoxygenase activity (+).The data is presented as percent inhibition of assays without inhibitorvs. inhibitor concentration (μg/mL). The IC₅₀ for 5-LO was 1.38μg/mL/unit of enzyme.

FIG. 11 depicts graphically a profile of the inhibition of COX-1 andCOX-2 by the Univestin™ composition produced through combination of theextracts of Free-B-ring flavonoids and flavans in a ratio of 85:15 asdescribed in Example 14. Univestin™ was examined for its inhibition ofthe peroxidase activity of recombinant ovine COX-1 (♦) and ovine COX-2(▪). The data is presented as percent inhibition of assays withoutinhibitor vs. inhibitor concentration (μg/mL). The IC₅₀ for COX-1 was0.76 μg/mL/unit of enzyme while the IC₅₀ for COX-2 was 0.80 μg/mL/unit.

FIG. 12 depicts graphically a profile of the inhibition of COX-1 andCOX-2 by the Univestin™ composition produced through combination ofFree-B-ring flavonoids and flavans extracts in a ratio of 50:50 asdescribed in Example 14. Univestin™ was examined for its inhibition ofthe peroxidase activity of recombinant ovine COX-1 (♦) and ovine COX-2(▪). The data is presented as percent inhibition vs. inhibitorconcentration (μg/mL). The IC₅₀ for COX-1 was 0.38 μg/mL/unit of enzymewhile the IC₅₀ for COX-2 was 0.84 μg/mL/unit.

FIG. 13 depicts graphically a profile of the inhibition of COX-1 andCOX-2 by the Univestin™ composition produced through combinationextracts of Free-B-ring flavonoids and flavans in a ratio of 20:80 asdescribed in Example 14. Univestin™ was examined for its inhibition ofthe peroxidase activity of recombinant ovine COX-1 (♦) and ovine COX-2(▪). The data is presented as percent inhibition of assays withoutinhibitor vs. inhibitor concentration (μg/mL). The IC₅₀ for COX-1 was0.18 μg/mL/unit of enzyme while the IC₅₀ for COX-2 was 0.41 μg/mL/unit.

FIG. 14 depicts the effect of increasing concentrations of Univestin™ onthe amount of LPS-induced newly synthesized LTB₄ (♦) as determined byELISA in THP-1 or HT-29 cells (ATCC). The activity of the combinationextract is expressed as % inhibition of induced LTB₄ synthesis.

FIG. 15 compares the LTB₄ levels as determined by ELISA that remain inHT-29 cells after treatment with 3 μg/mL Univestin™ in non-induced cellsto treatment with 3 μg/mL ibuprofen as described in Example 16.

FIG. 16 compares the effect of various concentrations of Univestin™ oncox-1 and cox-2 gene expression. The expression levels are standardizedto 18S rRNA expression levels (internal control) and then normalized tothe no-treatment, no-LPS condition. This Figure demonstrates a decreasein cox-2, but not cox-1 gene expression following LPS-stimulation andexposure to Univestin™.

FIG. 17 compares the effect of 3 μg/mL Univestin™ on cox-1 and cox-2gene expression with the equivalent concentration of other NSAIDs. Theexpression levels are standardized to 18S rRNA expression levels(internal control) and then normalized to the no-treatment, no-LPScondition.

FIG. 18 illustrates graphically ear-swelling data as a measure ofinhibition of inflammation. Univestin™ produced through the combinationof standardized extracts of Free-B-ring flavonoids and flavans in aratio of 80:20 was compared to untreated mice and mice givenindomethacin (50 mg/kg) via oral gavage. The data is presented as thedifference in micron measurement of the untreated vs. the treated earlobe for each mouse.

FIG. 19 shows the effect of 100 mg/kg of Univestin™ (80:20) ratio ofstandardized extracts of Free-B-ring flavonoids to flavans) on the AAinjected ankles of mice (Univestin™+arachidonic acid) compared tonon-treated mice (no treatment+arachidonic acid), mice without AAinjections (negative control) or mice that were injected with the liquidcarrier (vehicle control).

FIG. 20 illustrates graphically the 95% confidence interval for the painindex WOMAC score at baseline, 30, 60 and 90 days of treatment withUnivestin™ at a dosage of 250 mg/day.

FIG. 21 illustrates graphically the 95% confidence interval for the painindex WOMAC score at baseline, 30, 60 and 90 days of treatment withUnivestin™ at a dosage of 500 mg/day.

FIG. 22 illustrates graphically the 95% confidence interval for the painindex WOMAC score at baseline, 30, 60 and 90 days of treatment withcelecoxib at a dosage of 200 mg/day.

FIG. 23 illustrates graphically the 95% confidence interval for the painindex WOMAC score at baseline, 30, 60 and 90 days of treatment with theplacebo.

FIG. 24 illustrates graphically the 95% confidence interval for thestiffness index WOMAC score at baseline, 30, 60 and 90 days of treatmentwith Univestin™ at a dosage of 250 mg/day.

FIG. 25 illustrates graphically the 95% confidence interval for thestiffness index WOMAC score at baseline, 30, 60 and 90 days of treatmentwith Univestin™ at a dosage of 500 mg/day.

FIG. 26 illustrates graphically the 95% confidence interval for thestiffness index WOMAC score at baseline, 30, 60 and 90 days of treatmentwith celecoxib at a dosage of 200 mg/day.

FIG. 27 illustrates graphically the 95% confidence interval for thestiffness index WOMAC score at baseline, 30, 60 and 90 days of treatmentwith the placebo.

FIG. 28 illustrates graphically the 95% confidence interval for thefunctional impairment index WOMAC score at baseline, 30, 60 and 90 daysof treatment with Univestin™ at a dosage of 250 mg/day.

FIG. 29 illustrates graphically the 95% confidence interval for thefunctional impairment index WOMAC score at baseline, 30, 60 and 90 daysof treatment with Univestin™ at a dosage of 500 mg/day.

FIG. 30 illustrates graphically the 95% confidence interval for thefunctional impairment index WOMAC score at baseline, 30, 60 and 90 daysof treatment with celecoxib at a dosage of 200 mg/day.

FIG. 31 illustrates graphically the 95% confidence interval for thefunctional impairment index WOMAC score at baseline, 30, 60 and 90 daysof treatment with the placebo.

FIG. 32 shows the effect of Univestin™ at doses of 250 and 500 mg/day ondecreasing BMI compared to celecoxib at 200 mg/day and the placebo.

FIG. 33 shows the effect of Univestin™ at doses of 250 and 500 mg/day ondecreasing weight compared to celecoxib at 200 mg/day and the placebo.

FIG. 34 shows the effect of Univestin™ at doses of 250 and 500 mg/day onlowering blood glucose compared to placebo.

DETAILED DESCRIPTION OF THE INVENTION

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, a flavonoid refers to one or moreflavonoids. As such, the terms “a” or “an”, “one or more” and “at leastone” are used interchangeably herein.

“Free-B-ring Flavonoids” as used herein are a specific class offlavonoids, which have no substitute groups on the aromatic B ring, asillustrated by the following general structure:

wherein

R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

“Flavans” are a specific class of flavonoids, which can be generallyrepresented by the following general structure:

wherein

R¹, R₂, R₃, R₄ and R₅ are independently selected from the groupconsisting of H, —OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂,—NR₃ ⁺X⁻, esters of substitution groups, including, but not limited to,gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters,trihydroxybenzoyl esters and caffeoyl esters and their chemicalderivatives thereof; carbon, oxygen, nitrogen or sulfur glycoside of asingle or a combination of multiple sugars including, but not limitedto, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof; dimer, trimer and other polymerizedflavans;

wherein

R is an alkyl group having between 1-10 carbon atoms; and

X is selected from the group of pharmaceutically acceptable counteranions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

“Gene expression” refers to the transcription of a gene to mRNA.

“Protein expression” refers to the translation of mRNA to a protein.

“RT-qPCR” is a method for reverse transcribing (RT) an mRNA moleculeinto a cDNA molecule and then quantitatively evaluating the level ofgene expression using a polymerase chain reaction (PCR) coupled with afluorescent reporter.

“Therapeutic” as used herein, includes treatment and/or prophylaxis.When used, therapeutic refers to humans as well as other animals.

“Pharmaceutically or therapeutically effective dose or amount” refers toa dosage level sufficient to induce a desired biological result. Thatresult may be the alleviation of the signs, symptoms or causes of adisease or any other alteration of a biological system that is desired.

“Placebo” refers to the substitution of the pharmaceutically ortherapeutically effective dose or amount sufficient to induce a desiredbiological that may alleviate the signs, symptoms or causes of a diseasewith a non-active substance.

A “host” or “patient” is a living subject, human or animal, into whichthe compositions described herein are administered.

Note that throughout this application various citations are provided.Each citation is specifically incorporated herein in its entirety byreference.

The present invention includes a novel composition of matter comprisedof a mixture of Free-B-ring flavonoids and flavans. This novelcomposition of matter is referred to herein as Univestin™. The ratio ofFree-B-ring flavonoids to flavans in the composition of matter can beadjusted based on the indications and the specific requirements withrespect to prevention and treatment of a specific disease or condition.Generally, the ratio of Free-B-ring flavonoids to flavans can be in therange of 99:1 Free-B-ring flavonoids:flavans to 1:99 of Free-B-ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodimentthe Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

In one embodiment of the present invention, the standardized Free-B-ringflavonoid extract is comprised of the active compounds with a purity ofbetween 1-99% (by weight) of total Free-B-ring flavonoids as defined inexamples 5, 7 and 13; Tables 5, 7, 8 and 9 and FIG. 3. Baicalin is themajor active component in the extract, which accounts for approximately50-90% (by weight) of the total Free-B-ring flavonoids. In a preferredembodiment, the standardized extract contains >70% total Free-B-ringflavonoids in which >75% of the Free-B-ring flavonoids is baicalin.

In one embodiment, the standardized flavan extract is comprised of theactive compounds with a purity of between 1-99% (by weight) totalflavans as defined in Example 8, 9 and 12; Tables 4, 6 and 9 and FIG. 9.Catechin is the major active component in the extract and accounts for50-90% (by weight) of the total flavans. In a preferred embodiment, thestandardized flavan extract contains >50% total flavans in which >70% offlavans is catechin.

In one embodiment Univestin™ is be produced by mixing the above twoextracts or synthetic compounds in a ratio from 99:1 to 1:99. Thepreferred ratio of Free-B-ring flavonoids to flavans is 85:15Free-B-ring flavonoids:flavans as defined in Example 14.

The concentration of Free-B-ring flavonoids in Univestin™ can be fromabout 1% to 99% and the concentration of flavans in Univestin™ can befrom 99% to 1%. In a preferred embodiment of the invention, theconcentration of total Free-B-ring flavonoids in Univestin™ isapproximately 75% with a baicalin content of approximately 60% of totalweight of the Univestin™; and the concentration of total flavans inUnivestin™ is approximately 10% with a catechin content of approximately9%. In this embodiment, the total active components (Free-B-ringflavonoids plus flavans) in Univestin™ are >80% of the total weight.

The present invention also includes methods that are effective insimultaneously inhibiting both the COX-2 and 5-LO enzymes. The methodfor the simultaneous dual inhibition of the COX-2 and 5-LO pathways iscomprised of administering a composition comprising a mixture ofFree-B-ring flavonoids and flavans synthesized and/or isolated from asingle plant or multiple plants to a host in need thereof. The ratio ofFree-B-ring flavonoids to flavans in the composition can be in the rangeof 99:1 Free-B-ring flavonoids:flavans to 1:99 of Free-B-ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodiment,the Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

The present further includes methods for the prevention and treatment ofCOX-2 and 5-LO mediated diseases and conditions. The method forpreventing and treating COX-2 and 5-LO mediated diseases and conditionsis comprised of administering to a host in need thereof an effectiveamount of a composition comprising a mixture of Free-B-ring flavonoidsand flavans synthesized and/or isolated from a single plant or multipleplants together with a pharmaceutically acceptable carrier. The ratio ofFree-B-ring flavonoids to flavans can be in the range of 99:1Free-B-ring flavonoids:flavans to 1:99 of Free-B-ringflavonoids:flavans. In specific embodiments of the present invention,the ratio of Free-B-ring flavonoids to flavans is selected from thegroup consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodiment,the Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

In yet a further embodiment, the present includes a method for treatinggeneral joint pain and stiffness, improving mobility and physicalfunction and preventing and treating pathological conditions ofosteoarthritis and rheumatoid arthritis. The method for preventing andtreating joint pain and stiffness, improving mobility and physicalfunction and preventing and treating pathological conditions ofosteoarthritis, and rheumatoid arthritis is comprised of byadministering to a host in need thereof an effective amount of acomposition comprising a mixture of Free-B-ring flavonoids and flavanssynthesized and/or isolated from a single plant or multiple plantstogether with a pharmaceutically acceptable carrier. The ratio ofFree-B-ring flavonoids to flavans can be in the range of 99:1 to 1:99Free-B-ring flavonoids:flavans. In specific embodiments of the presentinvention, the ratio of Free-B-ring flavonoids to flavans is selectedfrom the group consisting of approximately 90:10, 80:20, 70:30, 60:40,50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodiment,the Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

The present invention also includes a method for modulating theproduction of mRNA implicated in pain pathways said method comprisingadministering to a host in need thereof an effective amount of acomposition comprising a mixture of Free-B-ring flavonoids and flavanssynthesized and/or isolated from a single plant or multiple plants andoptionally a pharmaceutically acceptable carrier. The ratio ofFree-B-ring flavonoids to flavans can be in the range of 99:1 to 1:99Free-B-ring flavonoids:flavans. In specific embodiments of the presentinvention, the ratio of Free-B-ring flavonoids to flavans is selectedfrom the group consisting of approximately 90:10, 80:20, 70:30, 60:40,50:50, 40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of theinvention, the ratio of Free-B-ring flavonoids:flavans in thecomposition of matter is approximately 85:15. In a preferred embodimentthe Free-B-ring flavonoids are isolated from a plant or plants in theScutellaria genus of plants and flavans are isolated from a plant orplants in the Acacia genus of plants.

The Free-B-ring flavonoids that can be used in accordance with themethod of this invention include compounds illustrated by the generalstructure set forth above. The Free-B-ring flavonoids of this inventionmay be obtained by synthetic methods or may be isolated from the familyof plants including, but not limited to Annonaceae, Asteraceae,Bignoniaceae, Combretaceae, Compositae, Euphorbiaceae, Labiatae,Lauranceae, Leguminosae, Moraceae, Pinaceae, Pteridaceae,Sinopteridaceae, Ulmaceae, and Zingiberaceae. The Free-B-ring flavonoidscan also be extracted, concentrated, and purified from the genera ofhigh plants, including but not limited to Desmos, Achyrocline, Oroxylum,Buchenavia, Anaphalis, Cotula, Gnaphalium, Helichrysum, Centaurea,Eupatorium, Baccharis, Sapium, Scutellaria, Molsa, Colebrookea, Stachys,Origanum, Ziziphora, Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza,Millettia, Pongamia, Tephrosia, Artocarpus, Ficus, Pityrogramma,Notholaena, Pinus, Ulmus, and Alpinia.

The Free-B-ring flavonoids can be found in different parts of plants,including but not limited to stems, stem barks, twigs, tubers, roots,root barks, young shoots, seeds, rhizomes, flowers and otherreproductive organs, leaves and other aerial parts. Methods for theisolation and purification of Free-B-ring flavonoids are described inU.S. application Ser. No. 10/091,362, filed Mar. 1, 2002, entitled“Identification of Free-B-ring Flavonoids as Potent COX-2 Inhibitors,”which is incorporated herein by reference in its entirety.

The flavans that can be used in accordance with the method of thisinvention include compounds illustrated by the general structure setforth above. The flavans of this invention may be obtained by syntheticmethods or may be isolated from a plant or plants selected from theAcacia genus of plants. In a preferred embodiment, the plant is selectedfrom the group consisting of Acacia catechu, A. concinna, A. farnesiana,A. Senegal, A. speciosa, A. arabica, A. caesia, A. pennata, A. sinuata.A. mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A.holoserecia and A. mangium.

The flavans can be found in different parts of plants, including but notlimited to stems, stem barks, trunks, trunk barks, twigs, tubers, roots,root barks, young shoots, seeds, rhizomes, flowers and otherreproductive organs, leaves and other aerial parts. Methods for theisolation and purification of flavans are described in U.S. applicationSer. No. 10/104,477, filed Mar. 22, 2002, entitled “Isolation of a DualCOX-2 and 5-Lipoxygenase Inhibitor from Acacia,” which is incorporatedherein by reference in its entirety.

The present invention implements a strategy that combines a series of invivo studies as well as in vitro biochemical, cellular, and geneexpression screens to identify active plant extracts and components thatspecifically inhibit COX-2 and 5-LO enzymatic activity, and impactcox-2, but not cox-1 mRNA production. The methods used herein toidentify active plant extracts and components that specifically inhibitCOX-2 and 5-LO pathways are described in Examples 1 to 13 (FIGS. 1-10).These methods are described in greater detail in U.S. application Ser.No. 10/091,362, filed Mar. 1, 2002, entitled “Identification ofFree-B-ring Flavonoids as Potent COX-2 Inhibitors” and U.S. applicationSer. No. 10/104,477, filed Mar. 22, 2002, entitled “Isolation of a DualCOX-2 and 5-Lipoxygenase Inhibitor from Acacia,” each of which isspecifically incorporated herein by reference in its entirety.

These studies resulted in the discovery of a novel composition of matterreferred to herein as Univestin™, which is comprised of a proprietaryblending of two standardized extracts, which contain Free-B-ringflavonoids and flavans, respectively. A general example for preparingsuch a composition is provided in Example 14 using two standardizedextracts isolated from Acacia and Scutellaria, respectively, togetherwith one or more excipients. The Acacia extract used in Example 14contained >60% total flavans, as catechin and epicatechin, and theScutellaria extract contained >70% Free-B-ring flavonoids, which wasprimarily baicalin. The Scutellaria extract contained other minoramounts of Free-B-ring flavonoids as set forth in Table 11. One or moreexcipients are optionally added to the composition of matter. The amountof excipient added can be adjusted based on the actual active content ofeach ingredient desired. A blending table for each individual batch ofproduct must be generated based on the product specification and QCresults for individual batch of ingredients. Additional amounts ofactive ingredients in the range of 2-5% are recommended to meet theproduct specification. Example 14 illustrates a blending table that wasgenerated for one batch of Univestin™ (Lot#G1702-COX-2). Differentblending ratios of the formulated Univestin™ product were tested fortheir ability to inhibit COX-2 and 5-LO enzyme activities, and to reducecox mRNA production as described in Examples 15-17.

The COX-2 inhibition assay relied on the activity of the enzymeperoxidase in the presence of heme and arachidonic acid. In order toscreen for compounds that inhibited COX-1 and COX-2 activity, a highthroughput, in vitro assay was developed that utilized inhibition of theperoxidase activity of both enzymes as illustrated in Examples 2 and 6.After isolating plant fractions that inhibited COX-2 activity in thescreening process, the two individual standardized extracts, onecomposed primarily of Free-B-ring flavonoids (isolated from Scutellaria)and the other of flavans (isolated from Acacia), were compared, as wellas, purified components from each extract and different ratios of thecombined extracts by titrating against a fixed amount of the COX-1 andCOX-2 enzymes. This study revealed that the purified Free-B-ringflavonoids, baicalin and baicalein isolated from Scutellaria baicalensisand the purified flavan, catechin isolated from Acacia catechu,inhibited COX-2 and 5-LO activity. Additionally, each of the individualstandardized extracts, which contained concentrations of Free-B-ringflavonoids in the range of 10-90% (based on HPLC) and flavans in therange of 10-90% (based on HPLC), also inhibited COX-2 and 5-LO activity.Finally, the study revealed that compositions containing mixtures ofeach of the individual standardized extracts having ratios ofFree-B-ring flavonoids to flavans of approximately 80:20, 50:50, and20:80, were also all highly effective at inhibiting COX-2 enzymaticactivity in vitro. The results are set forth in FIGS. 11-13).

Example 16 describes cell assays performed that targeted inhibition ofcompounds in the breakdown of arachidonic acid in the 5-LO pathway,namely LTB₄. The results are set forth in FIGS. 14 and 15.

Example 17 describes an experiment performed to determine differentialinhibition of the cox-2 gene by Univestin™. Gene expression data wasobtained for the inhibition of cox-1 and cox-2 mRNA production in asemi-quantitative RT-qPCR assay. The results are set forth in FIGS. 16and 17. With reference to FIG. 16 it can be seen that Univestin™inhibited cox-2 mRNA production without effecting cox-1 gene expression.In addition, when compared with other COX-2 inhibitor drugs, Univestin™was able to decrease LPS-stimulated increases in cox-1 and cox-2 geneexpression. Importantly, celecoxib and ibuprofen both increased cox-2gene expression (FIG. 17).

In vivo efficacy was demonstrated by the application of skin irritatingsubstances, such as AA, to the ears of mice and measuring the reductionof swelling in mice treated with Univestin™ as described in Example 18.The results are set forth in FIG. 18. Additionally, efficacy at the siteof inflammation and pain, was determined by the injection of an irritantinto the ankle joints of mice and measuring the reduction of swelling inmice treated with Univestin™, as described in Example 19. The resultsare set forth in FIG. 19.

Individual standardized extracts containing concentrations ofFree-B-ring flavonoids in the range of 10-99% (based on HPLC) andflavans in the range of 10-99% (based on HPLC) as well as the productUnivestin™ were tested for toxicity in mice with chronic and acuteadministration (data not shown). In the chronic administration protocol,mice were fed the test articles by oral gavage with daily doses of 90mg/kg (equivalent to the human daily dose of 500 mg), 450 mg/kg (fivetimes the daily-dose equivalent) and 900 mg/kg (ten times the daily-doseequivalent). Mice showed no adverse effects in terms of weight gain,physical appearance or behavior. Gross necropsy results showed no organabnormalities and histology of the stomach, kidney, and liver showed nodifferences compared to untreated control mice. Full blood workmeasuring electrolytes, blood proteins, blood enzymes, and liver enzymesshowed no abnormalities compared to the untreated control mice. In theacute protocol, individual standardized extracts containingconcentrations of Free-B-ring flavonoids in the range of 10-99% (basedon HPLC) and flavans in the range of 10-99% (based on HPLC) as well asthe product Univestin™ given at 2 grams/kg (20 times the daily-doseequivalent) showed no abnormalities in weight gain, appearance,behavior, gross necropsy appearance of organs, histology of stomach,kidney, and liver or blood work.

Example 20 describes a clinical study performed to evaluate the efficacyof Univestin™ on the relief of pain caused by rheumatoid arthritis orosteoarthritis of the knee and/or hip. The study was a single-center,randomized, double-blind, placebo-controlled study. Sixty subjects(n=60) with rheumatoid arthritis or osteoarthritis of the knee and/orhip were randomly placed into four groups and treated for 90 days with aplacebo, Univestin™ (250 mg/day or 500 mg/day) or Celebrex™ (also knownas celecoxib) (200 mg/day). The Univestin™, as illustrated in Example14, Table 11, consisted of a proprietary blend of standardized extractof Scutellaria baicalensis Georgi with a baicalin content of 82.2% (w/w)and total Free-B-ring Flavonoids >90% (w/w) and a standardized extractof Acacia catechu with a total flavan content of 77.2% (w/w) in a ratioof 85:15. Celebrex™ is a trade name for a prescription drug that is aCOX-2 selective inhibitor. Table 12 sets forth the WOMAC index scoresfor pain, stiffness and function before treatment (baseline scores) andat 30, 60 and 90 days. Table 13 sets forth the absolute changes in WOMACindex scores for pain, stiffness and function after treatment for 30, 60and 90 days. FIGS. 20-31 illustrate the results of this studygraphically plotting the 95% confidence intervals for all data.

As shown in the FIGS. 20 to 31, the WOMAC composite scores andindividual subscores, related to pain, stiffness and physical functionexhibited significant improvements during administration of Univestin™compared to the placebo group. Further, Univestin™ exhibited a similareffectiveness on pain relieve, better effectiveness at decreasingstiffness, and marked improvement of physical function compared to theprescription drug Celebrex™. The greatest significance can be seen incomparing each dose of Univestin™ to the placebo and celecoxib inrelieving pain, stiffness and functional impairment associated withosteoarthritis or rheumatoid arthritis.

Multiple post-hoc comparisons for each treatment group pairs within theAnalysis of Variance models showed that Univestin™ at 500 mg/day wassignificantly more effective than celecoxib at 200 mg/day for thereduction of pain caused osteoarthritis during the 30 days (p=0.020) oftreatment. In addition, the administration of a dose of 500 mg/day ofUnivestin™ was also significantly more effective than the placebo forthe reduction of pain within 30 days (p=0.044), 60 days (p=0.032) and 90days (p=0.001) of treatment. Celecoxib at 200 mg/day showed significancefor the reduction of pain vs. the placebo at 60 days (p=0.009) oftreatment. At 90 days, the 500 mg/day Univestin™ dose showedsignificantly higher effectiveness compared to the 250 mg/day dosewithin 90 days (p=0.038) of treatment.

Univestin™ at 250 mg/day was significantly more effective than theplacebo for the reduction of stiffness caused by osteoarthritis, within30 days (p=0.00), 60 days (p=0.027) and 90 days (p=0.015) of treatment.In addition, Univestin™ at a dose of 500 mg/day was significantly moreeffective than placebo for reduction of stiffness caused byosteoarthritis, within 30 days (p=0.001) and 90 days (p=0.005) oftreatment. Celecoxib at 200 mg/day showed significantly moreeffectiveness than the placebo for the reduction of stiffness caused byosteoarthritis only at 30 days (p=0.023) of treatment.

For reduction of functional impairment caused by osteoarthritis,Univestin™ was significantly more effective than celecoxib at 200 mg/daywithin 30 days (p=0.010) of treatment. In addition, the 250 mg/day doseof Univestin™ was also significantly more effective than placebo for thereduction of functional impairment caused by osteoarthritis within 30days (p=0.010), 60 days (p=0.043) and 90 days (p=0.039) of treatment.The 500 mg/day dose of Univestin™ was more effective than celecoxib at200 mg/day within 30 days (p=0.015), 60 days (p=0.043) and 90 days(0.039) of treatment. Finally, the 500 mg/day dose of Univestin™ wasalso significantly more effective than placebo for the reduction offunctional impairment caused by osteoarthritis within 30 days (p=0.015),60 days (p=0.016) and 90 days (p=0.003) of treatment.

These results suggest that Univestin™, particularly at a dosage of 500mg/day, is much more effective than the placebo and celecoxib atrelieving pain, stiffness and improving functional impairment caused byosteoarthritis. Additionally, Univestin™ administered at a dosage of 250mg/day is also very effective at relieving stiffness and improvingfunctional impairment caused by osteoarthritis compared to the placeboand celecoxib. Celecoxib also showed only marginal improvement overallin relieving pain, stiffness and functional impairment caused byosteoarthritis.

In addition to the effects of Univestin™ on pain, stiffness andfunctional impairment caused by osteoarthritis, Example 21 shows ameasurable effect by Univesti™ on body mass index (BMI) and weight loss.While not limited by theory, this effect may be due to an increase inmobility as a result of the administration of an anti-inflammatory ormay also be due to a specific mechanism that increases metabolism orreduces the utilization of fats and carbohydrates in the body. Table 14shows the effect of Univestin™ administered at a dose of 250 and 500mg/day as well as celecoxib and placebo on weight and BMI after 30 and90 days of treatment. The results are illustrated graphically in FIGS.32 and 33. With reference to FIGS. 32 and 33, it can be seen thatUnivestin™ administered at a dosage of both 250 and 500 mg/day resultedin a significant drop in weight and BMI after thirty days, with weightloss almost doubling after 90 days. Celecoxib had a smaller effect onweight and BMI as compared to Univestin™.

Multiple post-hoc comparisons for each treatment group pairs with theAnalysis of Variance models were also performed for weight loss and BMIas described in Example 21. These analyses showed that Univestin™ at 250mg/day and 500 mg/day doses caused statistically significant weight loss(p=0.011 vs. p=0.118) against the placebo after 30 days of treatment.Celecoxib did not cause significant weight loss against placebo at 30days. The weight loss continued throughout 90 days of treatment withUnivestin™ at 250 and 500 mg/day with statistical significance versusplacebo (p=0.001 and 0.01 receptively). Celecoxib still did not showsignificance relative to the placebo. The decrease of BMI followedsimilar trends for the 250 mg/day dose of Univestin™ which wassignificant relative to the placebo after 30 days (p=0.008) as well as90 days (p=0.001). The 500 mg/day dose of Univestin™ showed decreasingof BMI without statistical significance at 30 days of treatment.However, the decrease of BMI reached statistical significance 90 days oftreatment (p=0.011). Again, after 90 days of treatment, the celecoxibtreatment group showed no statistically significant changes in BMIversus placebo.

Example 22 suggests that administration of Univestin™ may affect bloodglucose levels as well as its effect on weight loss and BMI. Measurabledifferences in blood glucose levels are detected with 30 days ofinitiating treatment with Univestin™. At 90 days, both the 250 and 500mg/day Univestin™ treated groups showed significant drops in bloodglucose levels. The effect of celecoxib on blood glucose was lessdramatic. The results are set forth in Table 15 and illustratedgraphically in FIG. 34.

Once again multiple post-hoc comparisons for each treatment group pairswith the Analysis of Variance models were also performed for bloodglucose as described in Example 22. Only the 500 mg/day dose ofUnivestin™ showed statistically relevant significance versus the placebogroup (after 30 days, p=0.028; after 90 days, p=0.022). The 250 mg/daydose of Univestin™, however, showed clinically significant changes inblood glucose levels versus the placebo.

The applicant believes that U.S. application Ser. No. 10/104,477, filedMar. 22, 2002, entitled “Isolation of a Dual COX-2 and 5-LipoxygenaseInhibitor from Acacia,” is the first report of a composition of matterisolated from the Acacia genus of plants that demonstrates dualspecificity for COX-2 and 5-LO and that U.S. application Ser. No.10/091,362, filed Mar. 1, 2002, entitled “Identification of Free-B-ringFlavonoids as Potent COX-2 Inhibitors,” is the first report of acorrelation between Free-B-ring flavonoid structure and COX-2 inhibitoryactivity. These discoveries led to a novel blending of two classes ofspecific compounds—Free-B-ring flavonoids and flavans—to produce acomposition of matter, referred to herein as Univestin™, which can beused for alleviating joint pain and stiffness, improving mobility andphysical function and preventing and treating the pathologicalconditions of osteoarthritis, and rheumatoid arthritis.

While not limited by theory, the identified mechanism of action of thisformulation is believed to be the direct inhibition of both theperoxidase activity of the COX-2 enzyme and the 5-LO enzyme activity,together with a decrease in the mRNA production of each of theseenzymes. Univestin™ can also be utilized to prevent and treat COX-2 and5-LO mediated diseases and conditions, including, but are not limited toosteoarthritis, rheumatoid arthritis, menstrual cramps,arteriosclerosis, heart attack, obesity, diabetes, syndrome X,Alzheimer's disease, respiratory allergic reaction, chronic venousinsufficiency, hemorrhoids, Systemic Lupus Erythromatosis, psoriasis,chronic tension headache, migraine headaches, inflammatory bowl disease;topical infections caused by virus, bacteria, fungus, sunburn, thermalburns, contact dermatitis, melanoma and carcinoma. Finally, Univestin™has been found in human clinical study that it can cause weight loss andreduce blood glucose level due to improvement of flexibility, mobilityand increase physical activity.

The present invention is also directed toward therapeutic compositionscomprising the therapeutic agents of the present invention. Thetherapeutic agents of the instant invention can be administered by anysuitable means, including, for example, parenteral, topical, oral orlocal administration, such as intradermally, by injection, or byaerosol. The particular mode of administration will depend on thecondition to be treated. It is contemplated that administration of theagents of the present invention may be via any bodily fluid, or anytarget or any tissue accessible through a body fluid. In the preferredembodiment of the invention, the agent is administered by injection.Such injection can be locally administered to any affected area. Atherapeutic composition can be administered in a variety of unit dosageforms depending upon the method of administration. For example, unitdosage forms suitable for oral administration of an animal includepowder, tablets, pills and capsules. Preferred delivery methods for atherapeutic composition of the present invention include intravenousadministration and local administration by, for example, injection ortopical administration. A therapeutic reagent of the present inventioncan be administered to any animal, preferably to mammals, and morepreferably to humans.

For particular modes of delivery, a therapeutic composition of thepresent invention can be formulated so as to include other componentssuch as a pharmaceutically acceptable excipient, an adjuvant, and/or acarrier. For example, compositions of the present invention can beformulated in an excipient that the animal to be treated can tolerate.Examples of such excipients, include but are not limited to cellulose,silicon dioxide, dextrates, sucrose, sodium starch glycolate, calciumphosphate, calcium sulfate, water, saline, Ringer's solution, dextrosesolution, mannitol, Hank's solution, and other aqueous physiologicallybalanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesameoil, ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity-enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer, Tris buffer, histidine, citrate,and glycine, or mixtures thereof, while examples of preservativesinclude thimerosal, m- or o-cresol, formalin and benzyl alcohol.Standard formulations can either be liquid injectables or solids, whichcan be taken up in a suitable liquid as a suspension or solution forinjection. Thus, in a non-liquid formulation, the excipient can comprisedextrose, human serum albumin, preservatives, etc., to which sterilewater or saline can be added prior to administration.

In one embodiment of the present invention, the composition can alsoinclude an adjuvant or a carrier. Adjuvants are typically substancesthat generally enhance the function of the formula in preventing andtreating indications related to COX & LO pathways. Suitable adjuvantsinclude, but are not limited to, Freund's adjuvant; other bacterial cellwall components; aluminum-based salts; calcium-based salts; silica;boron, histidine, glucosamine sulfates, Chondroitin sulfate, coppergluconate, polynucleotides; vitamin D, vitamin K, toxoids; shark andbovine cartilage; serum proteins; viral coat proteins; otherbacterial-derived preparations; gamma interferon; block copolymeradjuvants, such as Hunter's Titermax adjuvant (Vaxcel™, Inc. Norcross,Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives, such as Quil A(available from Superfos Biosector A/S, Denmark). Carriers are typicallycompounds that increase the half-life of a therapeutic composition inthe treated animal. Suitable carriers include, but are not limited to,polymeric controlled release formulations, biodegradable implants,liposomes, bacteria, viruses, oils, esters, and glycols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

Once the therapeutic composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder; or directly capsulated and/or tabletedwith other inert carriers for oral administration. Such formulations maybe stored either in a ready to use form or requiring reconstitutionimmediately prior to administration. The manner of administeringformulations containing the compositions for systemic delivery may bevia oral, subcutaneous, intramuscular, intravenous, intranasal orvaginal or rectal suppository.

The amount of the composition that will be effective in the treatment ofa particular disorder or condition will depend on the nature of thedisorder of condition, which can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness or advancement of the disease orcondition, and should be decided according to the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curved derived from in vitro or animal model test systems.For example, an effective amount of the composition can readily bedetermined by administering graded doses of the composition andobserving the desired effect.

The method of treatment according to this invention comprisesadministering internally or topically to a patient in need thereof atherapeutically effective amount of the composition comprised of amixture of Free-B-ring flavonoids and flavans. The purity of the mixtureincludes, but is not limited to 0.01% to 100%, depending on themethodology used to obtain the compound(s). In a preferred embodiment,doses of the mixture of Free-B-ring flavonoids and flavans andpharmaceutical compositions containing the same are an efficacious,nontoxic quantity generally selected from the range of 0.01 to 200 mg/kgof body weight. Persons skilled in the art using routine clinicaltesting are able to determine optimum doses for the particular ailmentbeing treated.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES Example 1 Preparation of Organic and Aqueous Extracts fromAcacia and Scutellaria Plants

Plant material from Acacia catechu (L) Willd. barks, Scutellariaorthocalyx roots, Scutellaria baicalensis roots or Scutellarialateriflora whole plant was ground to a particle size of no larger than2 mm. Dried ground plant material (60 g) was then transferred to anErlenmeyer flask and methanol:dichloromethane (1:1) (600 mL) was added.The mixture was shaken for one hour, filtered and the biomass wasextracted again with methanol:dichloromethane (1:1) (600 mL). Theorganic extracts were combined and evaporated under vacuum to providethe organic extract (see Table 1 below). After organic extraction, thebiomass was air dried and extracted once with ultra pure water (600 mL).The aqueous solution was filtered and freeze-dried to provide theaqueous extract (see Table 1 below).

TABLE 1 Yield of Organic and Aqueous Extracts of Acacia and ScutellariaSpecies Organic Aqueous Plant Source Amount Extract Extract Acaciacatechu barks 60 g 27.2 g 10.8 g Scutellaria orthocalyx roots 60 g 4.04g 8.95 g Scutellaria baicalensis roots 60 g 9.18 g 7.18 g Scutellarialateriflora 60 g 6.54 g 4.08 g (whole plant)

Example 2 Inhibition of COX-2 and COX-1 Peroxidase Activity by PlantExtracts from Acacia Catechu, Various Scutellaria Species and OtherPlants

The bioassay directed screening process for the identification ofspecific COX-2 inhibitors was designed to assay the peroxidase activityof the enzyme as described below.

Peroxidase Assay.

The assay to detect inhibitors of COX-2 was modified for a highthroughput platform (Raz). Briefly, recombinant ovine COX-2 (Cayman) inperoxidase buffer (100 mM TBS, 5 mM EDTA, 1 μM Heme, 1 mg epinephrine,0.094% 10 phenol) was incubated with extract (1:500 dilution) for 15minutes. Quantablu (Pierce) substrate was added and allowed to developfor 45 minutes at 25° C. Luminescence was then read using a WallacVictor 2 plate reader. The results are presented in Table 2.

Table 2 sets forth the inhibition of enzyme by the organic and aqueousextracts obtained from five plant species, including the bark of Acaciacatechu, roots of two Scutellaria species and extracts from three otherplant species, which are comprised of structurally similar Free-B-ringflavonoids. Data is presented as the percent of peroxidase activityrelative to the recombinant ovine COX-2 enzyme and substrate alone. Thepercent inhibition by the organic extract ranged from 30% to 90%.

TABLE 2 Inhibition of COX-2 Peroxidase activity by various speciesInhibition of Inhibition of COX-2 by COX-2 by Plant Source organicextract aqueous extract Acacia catechu (bark) 75% 30% Scutellariaorthocalyx (root) 55% 77% Scutellaria baicalensis (root) 75% 0%Desmodium sambuense (whole plant) 55% 39% Eucaluptus globulus (leaf) 30%10% Murica nana (leaf) 90% 0%

Comparison of the relative inhibition of the COX-1 and COX-2 isoformsrequires the generation of IC₅₀ values for each of these enzymes. TheIC₅₀ is defined as the concentration at which 50% inhibition of enzymeactivity in relation to the control is achieved by a particularinhibitor. In these experiments, IC₅₀ values were found to range from 6to 50 μg/mL and 7 to 80 μg/mL for the COX-2 and COX-1 enzymes,respectively, as set forth in Table 3. Comparison of the IC₅₀ values ofCOX-2 and COX-1 demonstrates the specificity of the organic extractsfrom various plants for each of these enzymes. The organic extract ofScutellaria lateriflora for example, shows preferential inhibition ofCOX-2 over COX-1 with IC₅₀ values of 30 and 80 μg/mL, respectively.While some extracts demonstrate preferential inhibition of COX-2, othersdo not. Examination of the HTP fractions and purified compounds fromthese fractions is necessary to determine the true specificity ofinhibition for these extracts and compounds.

TABLE 3 IC₅₀ Values of Organic Extracts for Human and Ovine COX-2 andCOX-1 IC₅₀ IC₅₀ IC₅₀ Human Ovine Ovine COX-2 COX-2 COX-1 Plant Source(μg/mL) (μg/mL) (μg/mL) Acacia catechu (bark)  3 6.25 2.5 Scutellariaorthocalyx (root) Not done 10 10 Scutellaria baicalensis (root) 30 20 20Scutellaria lateriflora (whole plant) 20 30 80 Eucaluptus globulus(leaf) Not done 50 50 Murica nana (leaf)  5 6 7

Example 3 HTP Fractionation of Active Extracts

Organic extract (400 mg) from active plant was loaded onto a prepackedflash column. (2 cm ID×8.2 cm, 10 g silica gel). The column was elutedusing a Hitachi high throughput purification (HTP) system with agradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from100% A to 100% B in 30 minutes at a flow rate of 5 mL/min. Theseparation was monitored using a broadband wavelength UV detector andthe fractions were collected in a 96-deep-well plate at 1.9 mL/wellusing a Gilson fraction collector. The sample plate was dried under lowvacuum and centrifugation. DMSO (1.5 mL) was used to dissolve thesamples in each cell and a portion (100 μL was taken for the COXinhibition assay.

Aqueous extract (750 mg) from active plant was dissolved in water (5mL), filtered through a 1 μm syringe filter and transferred to a 4 mLHigh Pressure Liquid Chromatography (HPLC) vial. The solution was theninjected by an autosampler onto a prepacked reverse phase column (C-18,15 μm particle size, 2.5 cm ID×10 cm with precolumn insert). The columnwas eluted using a Hitachi high throughput purification (HTP) systemwith a gradient mobile phase of (A) water and (B) methanol from 100% Ato 100% B in 20 minutes, followed by 100% methanol for 5 minutes at aflow rate of 10 mL/min. The separation was monitored using a broadbandwavelength UV detector and the fractions were collected in a96-deep-well plate at 1.9 mL/well using a Gilson fraction collector. Thesample plate was freeze-dried. Ultra pure water (1.5 mL) was used todissolve samples in each cell and a portion (100 μL) was taken for theCOX inhibition assay.

Example 4 Inhibition of COX Peroxidase Activity by HTP Fractions fromAcacia and Scutellaria Species

Individual bioactive organic extracts were further characterized byexamining each of the HTP fractions for the ability to inhibit theperoxidase activity of both COX-1 and COX-2 recombinant enzymes. Theresults are presented in FIGS. 1 and 2, which depict the inhibition ofCOX-2 and COX-1 activity by HTP fractions from organic extracts of thebark of Acacia catechu and the roots of Scutellaria baicalensis isolatedas described in Examples 1 and 3 and assayed as described in Example 2.The profiles depicted in FIGS. 1 and 2 show multiple peaks of inhibitionthat indicate multiple active components in each extract. Several activepeaks are very selective for COX-2. Other Scutellaria sp. includingScutellaria orthocalyx and Scutellaria lateriflora demonstrate a similarpeak of inhibition (data not shown). However, both the COX-1 and COX-2enzymes demonstrate multiple peaks of inhibition suggesting that thereis more than one molecule contributing to the initial inhibitionprofiles.

Example 5 Isolation and Purification of the Active Free-B-RingFlavonoids from the Organic Extract of Scutellaria

The organic extract (5 g) from the roots of Scutellaria orthocalyx,isolated as described in Example 1, was loaded onto prepacked flashcolumn (120 g silica, 40 μm particle size 32-60 μm, 25 cm×4 cm) andeluted with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B)methanol from 100% A to 100% B in 60 minutes at a flow rate of 15mL/min. The fractions were collected in test tubes at 10 mL/fraction.The solvent was evaporated under vacuum and the sample in each fractionwas dissolved in 1 mL of DMSO and an aliquot of 20 μL was transferred toa 96 well shallow dish plate and tested for COX inhibitory activity.Based on the COX assay results, active fractions #31 to #39 werecombined and evaporated. Analysis by HPLC/PDA and LC/MS showed a majorcompound with a retention times of 8.9 minutes and a MS peak at 272 m/e.The product was further purified on a C18 semi-preparation column (25cm×1 cm), with a gradient mobile phase of (A) water and (B) methanol,over a period of 45 minutes at a flow rate of 5 mL/minute. Eighty-eightfractions were collected to yield 5.6 mg of light yellow solid. Puritywas determined by HPLC/PDA and LC/MS, and comparison with standards andNMR data. ¹H NMR: δ ppm. (DMSO-d6) 8.088 (2H, m, H-3′,5′), 7.577 (3H, m,H-2′,4′,6′), 6.932 (1H, s, H-8), 6.613 (1H, s, H-3). MS: [M+1]+=271 m/e.The compound was identified as baicalein. The IC₅₀ of baicalein againstthe COX-2 enzyme was determined to be 10 μg/mL.

Using preparative C-18 column chromatography, other Free-B-ringflavonoids were isolated and identified using a standardized extractisolated from the roots of Scutellaria baicalensis (lot # RM052302-01),having a Free-B-ring flavonoid content of 82.2%. Eleven structures wereelucidated using HPLC/PDA/MS as illustrated in FIG. 3. With reference toFIG. 3, the eleven compounds identified were baicalin,wogonin-7-glucuronide, oroxylin A 7-glucuronide, baicalein, wogonin,chrysin-7-glucuronide, 5-methyl-wogonin-7-glucuronide, scutellarin,norwogonin, chrysin and oroxylin A.

Example 6 COX Inhibition of Purified Free-B-Ring Flavonoids

Several Free-B-ring flavonoids have been obtained and tested at aconcentration of 20 μg/mL for COX-2 inhibitory activity using themethods described in Example 2. The results are summarized in Table 4.

Measurement of the IC₅₀ of baicalein, baicalin and a standardizedFree-B-ring flavonoid extract isolated from the roots of Scutellariabaicalensis was performed using the following method. A cleavable,peroxide chromophore was included in the assay to visualize theperoxidase activity of each enzyme in the presence of arachidonic acidas a cofactor. Typically, the assays were performed in a 96-well format.Each inhibitor, taken from a 10 mg/mL stock in 100% DMSO, was tested intriplicate at room temperature using the following range ofconcentrations: 0, 0.1, 1, 5, 10, 20, 50, 100, and 500 μg/mL. To eachwell, 150 μL of 100 mM Tris-HCl, pH 7.5 was added along with 10 μL of 22μM Hematin diluted in tris buffer, 10 μL of inhibitor diluted in DMSO,and 25 units of either COX-1 or COX-2 enzyme. The components were mixedfor 10 seconds on a rotating platform, after which 20 μL of 2 mMN,N,N′N′-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD) and 20 μLof 1.1 mM AA was added to initiate the reaction. The plate was shakenfor 10 seconds and then incubated for 5 minutes before reading theabsorbance at 570 nm. The inhibitor concentration vs. percentageinhibition was plotted and the IC₅₀ determined by taking thehalf-maximal point along the isotherm and intersecting the concentrationon the x-axis. The IC₅₀ was then normalized to the number of enzymeunits in the assay. The dose response and IC₅₀ results for baicalein,baicalin and a standardized Free-B-ring flavonoid extract isolated fromthe roots of Scutellaria baicalensis are provided in FIGS. 4, 5 and 6,respectively.

TABLE 4 Inhibition of COX Enzymatic Activity by Purified Free-B-ringFlavonoids Inhibition Inhibition Free-B-ring Flavonoids of COX-1 ofCOX-2 Baicalein 107% 109% 5,6-Dihydroxy-7-methoxyflavone 75% 59%7,8-Dihydroxyflavone 74% 63% Baicalin 95% 97% Wogonin 16% 12%

Example 7 HPLC Quantification of Free-B-Ring Flavonoids in ActiveExtracts Isolated from Scutellaria Orthocalyx (Roots), ScutellariaBaicalensis (Roots) and Oroxylum Indicum (Seeds)

The presence and quantity of Free-B-ring flavonoids in five activeextracts isolated from three different plant species have been confirmedand are set forth in the Table 5. The Free-B-ring flavonoids werequantitatively analyzed by HPLC using a Luna C-18 column (250×4.5 mm, 5μm) a using 1% phosphoric acid and acetonitrile gradient from 80% to 20%in 22 minutes. The Free-B-ring flavonoids were detected using a UVdetector at 254 nm and identified based on retention time by comparisonwith Free-B-ring flavonoid standards.

TABLE 5 Free-B-ring Flavonoid Content in Active Plant Extracts % TotalWeight Extractible amount of % Free-B-ring of from Free-B-ringFlavonoids in Active Extracts Extract BioMass Flavonoids Extract S.orthocalyx 8.95 g 14.9%  0.2 mg 0.6% (aqueous extract) S. orthocalyx3.43 g 5.7% 1.95 mg 6.4% (organic extract) S. baicalensis 7.18 g 12.0%0.03 mg 0.07%  (aqueous extract) S. baicalensis 9.18 g 15.3% 20.3 mg35.5%  (organic extract) Oroxylum indicum 6.58 g 11.0%  0.4 mg 2.2%(organic extract)

Example 8 Isolation and Purification of Active Compounds from theOrganic Extract of Acacia Catechu

The organic extract (5 g) from the roots of A. catechu, isolated asdescribed in Example 1, was loaded onto prepacked flash column (120 gsilica, 40 μm particle size 32-60 μm, 25 cm×4 cm) and eluted with agradient mobile phase of (A) 50:50 EtOAc:hexane and (B) methanol from100% A to 100% B in 60 minutes at a flow rate of 15 mL/min. Thefractions were collected in test tubes at 10 mL/fraction. The solventwas evaporated under vacuum and the sample in each fraction wasdissolved in DMSO (1 mL) and an aliquot of 20 μL was transferred to a 96well shallow dish plate and tested for COX inhibitory activity. Basedupon the COX assay results, active fractions #32 to #41 were combinedand evaporated to yield 2.6 g of solid. Analysis by HPLC/PDA and LC/MSshowed two major compounds with retention times of 15.8 and 16.1minutes, respectively. The product was further purified on a C18semi-preparatory column (25 cm×1 cm), loaded with 212.4 mg of productand eluted with a gradient mobile phase of (A) water and (B)acetonitrile (ACN), over a period of 60 minutes at a flow rate of 5mL/minute. Eighty-eight fractions were collected and two activecompounds were isolated. Compound 1 (11.5 mg) and Compound 2 (16.6 mg).Purity was determined by HPLC/PDA and LC/MS data by comparison withstandards (catechin and epicatechin) and NMR data.

Compound 1.

¹³C NMR: δ ppm (DMSO-d6) 27.84 (C4), 66.27 (C3), 80.96 (C2), 93.78 (C9),95.05 (C7), 99.00 (C5), 114.48 (C12), 115.01 (C15), 118.36 (C16), 130.55(C11), 144.79 (C14), 155.31 (C6), 156.12 (C10), 156.41 (C8). ¹H NMR: δppm. (DMSO-d6) 9.150 (1H, s, OH), 8.911 (1H, s, OH), 8.835 (1H, s, OH),8.788 (1H, s, OH), 6.706 (1H, d, J=2 Hz, H2′), 6.670 (1H, d, J=8.0 Hz,H-6′), 6.578 (1H, dd, J=2, 8 Hz, H-5′), 5.873 (1H, d, J=2 Hz, H8), 5.670(1H, d, J=2 Hz, H6), 4.839 (1H, d, J=4 Hz, OH), 4.461 (1H, d, J=7.3 Hz,H2), 3.798 (1H, m, H3), 2.625 (1H, m, H4b), 2.490 (1H, m, H4a). MS:[M+1]⁺=291 m/e. This compound was identified as catechin.

Compound 2.

¹³C NMR: δ ppm. (DMSO-d6) 28.17 (C4), 64.87 (C3), 78.02 (C2), 94.03(C9), 95.02 (C7), 98.44 (C5), 114.70 (C12), 114.85 (C15), 117.90 (C16),130.56 (C11), 144.39 (C14), 155.72 (C6), 156.19 (C10), 156.48 (C8). ¹HNMR: δ ppm. (DMSO-d6) 9.083 (1H, s, OH), 8.873 (1H, s, OH), 8.777 (1H,s, OH), 8.694 (1H, s, OH), 6.876 (1H, d, J=2 Hz, H2′), 6.646 (2H, s,H-5′, 6′), 5.876 (1H, d, J=2 Hz, H8), 5.700 (1H, d, J=2 Hz, H6), 4.718(1H, s, OH), 4.640 (1H, d, J=4.5 Hz, H2), 3.987 (1H, d, J=4.5 Hz, H3),2.663 (1H, dd, J=4.6, 6.3 Hz, H4b), 2.463 (1H, dd, J=4.6, 6.3 Hz, H4a).MS: [M+1]⁺=291 m/e. This compound was identified as epicatechin.

The dose response and IC₅₀ results for catechin and a standardizedflavan extract isolated from the bark of A. catechu are illustrated inFIGS. 7 and 8, using the method described in Example 6. The IC₅₀ valuesof epicatechin against the COX-1 and COX-2 enzymes are 7 μg/mL and 20μg/mL, respectively.

Example 9 HPLC Quantification of Active Extracts from Acacia Catechu

The flavan content in the organic and aqueous extracts isolated fromAcacia catechu were quantified by HPLC using a PhotoDiode Array detector(HPLC/PDA) and a Luna C18 column (250 mm×4.6 mm). The flavans wereeluted from the column using an acetonitrile gradient from 10% to 30%ACN over a period of 20 minutes, followed by 60% ACN for five minutes.The results are set forth in Table 6. A profile of the HPLC purificationis shown in FIG. 9. The flavans were quantified based on retention timeand PDA data using catechin and epicatechin as standards. The retentiontimes for the two major flavans were 12.73 minutes and 15.76 minutes,respectively.

TABLE 6 Free-B-ring Flavonoid Content in Active Plant Extracts ActiveExtracts from Weight of % Extractible % Flavans bark of A. catechuExtract from BioMass in Extract Aqueous Extract 10.8 g 18.0% 0.998%Organic Extract 27.2 g 45.3% 30.37%

Example 10 In Vitro Study of COX Inhibitory Activity of Organic Extractsfrom Acacia Catechu and Scutellaria Species

In vitro efficacy and COX-2 specificity of organic extracts isolatedfrom Acacia catechu and various Scutellaria species were tested incell-based systems for their ability to inhibit the generation of AAmetabolites. Cell lines HOSC, which constitutively express COX-2 andTHP-1, which express COX-1 were tested for their ability to generatePGE₂ in the presence of AA.

COX-2 Cell Based Assay.

HOSC (ATCC#8304-CRL) cells were cultured to 80-90% confluence. The cellswere trypsinized, washed and resuspended in 10 mL at 1×10⁶ cells/mL intissue culture media (MEM). The cell suspension (200 μL) was plated outin 96-well tissue culture plates and incubated for 2 hours at 37° C. and5% CO₂. The media was then replaced with new HOSC media containing 1ng/mL IL-1b and incubated overnight. The media was removed again andreplaced with 190 mL HOSC media. Test compounds were then added in 10 μLof HOSC media and were incubated for 15 minutes at 37° C. Arachidonicacid in HOSC media (20 mL, 100 μM) was added and the mixture wasincubated for 10 minutes on a shaker at room temperature. Supernatant(20 μL) was transferred to new plates containing 190 μL/well of 100 μMindomethacin in ELISA buffer. The supernatants were analyzed asdescribed below by ELISA.

COX-1 Cell Based Assay.

THP-1 cells were suspended to a volume of 30 mL (5×10⁵ cells/mL). TPAwas added to a final concentration of 10 nM TPA and cultured for 48hours to differentiate cells to macrophage (adherent). The cells wereresuspended in HBSS (25 mL) and added to 96-well plates in 200 mLvolumes at 5×10⁵ cells/well. The test compounds in RPMI 1640 (10 μL)were then added and incubated for 15 minutes at 37° C. Arachidonic acidin RPMI (20 μL) was then added and the mixture was incubated for 10minutes on a shaker at room temperature. Supernatant (20 μL) was addedto ELISA buffer (190 μL) containing indomethacin (100 μM). Thesupernatants were then analyzed by ELISA, as described below.

COX-2 Whole Blood Assay.

Peripheral blood from normal healthy donors was collected byvenipuncture. Whole blood (500 μL) was incubated with test compounds andextracts for 15 minutes at 37° C. Lipopolysaccharide (LPS, from E. coliserotype 0111:B4) was added to a final concentration of 100 μg/mL andcultured overnight at 37° C. The blood was centrifuged (12,000×g) andthe plasma was collected. Plasma (100 μL) was added to methanol (400 μL)to precipitate proteins. Supernatants were measured for PGE₂ productionby ELISA. This procedure is a modification of the methods described byBrideau et al. (1996) Inflamm. Res. 45:68-74.

COX-1 Whole Blood Assay.

Fresh blood was collected in tubes not containing anti-coagulants andimmediately aliquoted into 500 μL aliquots in siliconizedmicrocentrifuge tubes. Test samples were added, vortexed and allowed toclot for 1 hour at 37° C. The samples were then centrifuged (12,000×g)and the plasma was collected. The plasma (100 μL) was added to methanol(400 μL) to precipitate proteins. Supernatants were measured for TXB₂production by ELISA. This procedure is a modification of the methodsdescribed by Brideau et al. (1996) Inflamm. Res. 45:68-74.

ELISA Assays.

Immunolon-4 ELISA plates were coated with capture antibody 0.5-4 μg/mLin carbonate buffer (pH 9.2) overnight at 4° C. The plates were washedand incubated for 2 hours with blocking buffer (PBS+1% BSA) at roomtemperature. The plates were washed again and test sample (100 μL) wasadded and incubated for 1 hour at room temperature while shaking.Peroxidase conjugated secondary antibody was added in a 50 μL volumecontaining 0.5-4 mg/mL and incubated for 1 hour at room temperaturewhile shaking. The plates were then washed three times and TMB substrate(100 μL) was added. The plates were allowed to develop for 30 minutes,after which the reaction was stopped by the addition of 1 M phosphoricacid (100 μL). The plates were then read at 450 nm using a Wallac Victor2 plate reader.

Cytotoxicity.

Cellular cytotoxicity was assessed using a colorimetric kit (OxfordBiochemical Research) that measures the release of lactate dehydrogenasein damaged cells. Assays were completed according to manufacturer'sdirections. Both purified flavans and standardized extract from Acaciacatechu were tested. No cytotoxicity was observed for any of the testedcompounds.

The results of the assays are set forth in Table 7. The data arepresented as IC₅₀ values for direct comparison. With reference to Table5, IC₅₀ values are generally lower for COX-1 than COX-2. Additionally,whole blood was also measured for the differential inhibition of PGE₂generation (a measure of COX-2 in this system) or thromboxane B2 (TXB₂)(a measure of COX-1 activation). Referring to Table 7, these studiesclearly demonstrate specificity for COX-2 inhibition within the assaysbased on whole blood cells. However, studies using the THP-1 andHOSC-based model system actually showed greater selectivity for COX-1.Possible reasons for this discrepancy are the fundamental differencesbetween immortalized cell lines that constitutively express each of theenzymes and primary cells derived from whole blood that are induced toexpress COX enzymes. Primary cells are a more relevant model to studyinflammation in vivo. Additionally, the compounds used to identify COX-1vs. COX-2 activity vary in each of these systems and consequently arenot directly comparable.

TABLE 7 Inhibition of COX Activity in Cell Systems by Organic ExtractsCell Line Based Assay Whole Blood Assay Plant Source of the IC₅₀ IC₅₀IC₅₀ organic extracts IC₅₀ COX-2 COX-1 COX-2 COX-1 A. catechu (bark) 78μg/mL 22 μg/mL 40 μg/mL >50 μg/mL S. orthocalyx (root) 50 μg/mL 18 μg/mL10 μg/mL >50 μg/mL S. baicalensis (root) 82 μg/mL 40 μg/mL 20 μg/mL    8μg/mL S. lateriflora 60 μg/mL 30 μg/mL  8 μg/mL   20 μg/mL (whole plant)

Example 11 Inhibition of 5-Lipoxygenase by the Catechin from AcaciaCatechu

As noted above, one of the most important pathways involved in theinflammatory response is produced by non-heme, iron-containinglipoxygenases (5-LO, 12-LO, and 15-LO), which catalyze the addition ofmolecular oxygen onto fatty acids such as AA (AA) to produce thehydroperoxides 5-, 12- and 15-HPETE, which are then converted toleukotrienes. There were early indications that the flavan extract fromA. catechu may provide some degree of 5-LO inhibition, therebypreventing the formation of 5-HPETE. A Lipoxygenase Inhibitor ScreeningAssay Kit (Cayman Chemical, Inc., Cat#760700) was used to assess whetherthe purified flavan catechin from Acacia catechu directly inhibited 5-LOin vitro. The 15-LO from soybeans normally used in the kit was replacedwith potato 5-LO, after a buffer change from phosphate to a tris-basedbuffer using microfiltration was performed. This assay detects theformation of hydroperoxides through an oxygen sensing chromagen.Briefly, the assay was performed in triplicate by adding 90 μL of 0.17units/μL potato 5-LO, 20 μL of 1.1 mM AA, 100 μL of oxygen-sensingchromagen, and 10 μL of purified flavan inhibitor to finalconcentrations ranging from 0 to 500 μg/mL. The IC₅₀ for 5-LO inhibitionfrom catechin was determined to be 1.38 μg/mL/unit of enzyme.

Example 12 Preparation of a Standardized Extract from Acacia Catechu

Acacia catechu (500 mg of ground bark) was extracted with the followingsolvent systems. (1) 100% water, (2) 80:20 water:methanol, (3) 60:40water:methanol, (4) 40:60 water:methanol, (5) 20:80 water:methanol, (6)100% methanol, (7) 80:20 methanol:THF, (8) 60:40 methanol:THF. Theextracts were concentrated and dried under low vacuum. Theidentification of the chemical components in each extract was achievedby HPLC using a PhotoDiode Array detector (HPLC/PDA) and a 250 mm×4.6 mmC18 column. The chemical components were quantified based on retentiontime and PDA data using catechin and epicatechin as standards. Theresults are set forth in Table 8 and FIG. 9. As shown in Table 6, theflavan extract generated from solvent extraction with 80% methanol/waterprovided the best concentration of flavan components.

TABLE 8 Solvents for Generating Standardized Flavan Extracts from Acaciacatechu Total Extraction Weight of % Extractible amount of % CatechinsSolvent Extract from BioMass Catechins in Extract 100% water 292.8 mg58.56% 13 mg 12.02% water:methanol 282.9 mg 56.58% 13 mg 11.19% (80:20)water:methanol 287.6 mg 57.52% 15 mg 13.54% (60:40) water:methanol 264.8mg 52.96% 19 mg 13.70% (40:60) water:methanol 222.8 mg 44.56% 15 mg14.83% (20:80) 100% methanol 215.0 mg 43.00% 15 mg 12.73% methanol:THF264.4 mg 52.88% 11 mg 8.81% (80:20) methanol:THF 259.9 mg 51.98% 15 mg9.05% (60:40)

Example 13 Preparation of Standardized Free-B-Ring Flavonoid Extractsfrom Various Scutellaria Species

Scutellaria orthocalyx (500 mg of ground root) was extracted twice with25 mL of the following solvent systems. (1) 100% water, (2) 80:20water:methanol, (3) 60:40 water:methanol, (4) 40:60 water:methanol, (5)20:80 water:methanol, (6) 100% methanol, (7) 80:20 methanol:THF, (8)60:40 methanol:THF. The extracts were combined, concentrated and driedunder low vacuum. Identification of chemical components in each extractwas performed by HPLC using a PhotoDiode Array detector (HPLC/PDA) and a250 mm×4.6 mm C18 column. The chemical components were quantified basedon retention time and PDA data using baicalein, baicalin, scutellarein,and wogonin as standards. The results are set forth in Table 9.

TABLE 9 Quantification of Free-B-ring Flavonoids Extracted fromScutellaria orthocalyx Weight Total % Extraction of % Extractible amountof Flavonoids Solvent Extract from BioMass Flavonoids in Extract 100%water   96 mg 19.2% 0.02 mg 0.20% Water:methanol 138.3 mg 27.7% 0.38 mg0.38% (80:20) Water:methanol 169.5 mg 33.9% 0.78 mg 8.39% (60:40)Water:methanol 142.2 mg 28.4% 1.14 mg 11.26% (40:60) Water:methanol104.5 mg 20.9% 0.94 mg 7.99% (20:80) 100% methanol  57.5 mg 11.5% 0.99mg 10.42% methanol:THF  59.6 mg 11.9% 0.89 mg 8.76% (80:20) methanol:THF 58.8 mg 11.8% 1.10 mg 10.71% (60:40)

Scutellaria baicalensis (1000 mg of ground root) was extracted twiceusing 50 mL of a mixture of methanol and water as follows: (1) 100%water, (2) 70:30 water:methanol, (3) 50:50 water:methanol, (4) 30:70water:methanol, (5) 100% methanol. The extracts were combined,concentrated and dried under low vacuum. Identification of the chemicalcomponents was performed by HPLC using a PhotoDiode Array detector(HPLC/PDA), and a 250 mm×4.6 mm C18 column. The chemical components ineach extract were quantified based on retention time and PDA data usingbaicalein, baicalin, scutellarein, and wogonin standards. The resultsare set forth in Table 10.

TABLE 10 Quantification of Free-B-ring Flavonoids Extracted fromScutellaria baicalensis % Weight Extractible Total % Extraction of fromamount of Flavonoids Solvent Extract BioMass Flavonoids in Extract 100%water 277.5 mg 27.8%   1 mg 0.09% Water:methanol 338.6 mg 33.9% 1.19 mg11.48% (70:30) Water:methanol 304.3 mg 30.4% 1.99 mg 18.93% (50:50)Water:methanol 293.9 mg 29.4% 2.29 mg 19.61% (30:70) 100% methanol 204.2mg 20.4% 2.73 mg 24.51%

Example 14 Preparation of a Formulation with a Standardized Free-B-RingFlavonoid Extract from the Roots of Scutellaria Baicalensis and aStandardized Flavan Extract from the Bark of Acacia Catechu

A novel composition of matter, referred to herein as Univestin™ wasformulated using two standardized extracts isolated from Acacia andScutellaria, respectively, together with one or more excipients. Ageneral example for preparing such a composition is set forth below. TheAcacia extract used in this example contained >60% total flavans, ascatechin and epicatechin, and the Scutellaria extract contained >70%Free-B-ring flavonoids, which was primarily baicalin. The Scutellariaextract contained other minor amounts of Free-B-ring flavonoids as setforth in Table 11. One or more exipients is added to the composition ofmatter. The ratio of flavan and Free-B-ring flavonoids can be adjustedbased on the indications and the specific requirements with respect toinhibition of COX-2 vs. 5-LO and potency requirements of the product.The quantity of the excipients can be adjusted based on the actualactive content of each ingredient. A blending table for each individualbatch of product must be generated based on the product specificationand QC results for individual batch of ingredients. Additional amountsof active ingredients in the range of 2-5% are recommended to meet theproduct specification. Table 11 illustrates a blending table that wasgenerated for one batch of Univestin™ (Lot#G1702-COX-2).

Scutellaria baicalensis root extract (38.5 kg) (lot # RM052302-01)having a Free-B-ring flavonoid content of 82.2% (baicalin); Acaciacatechu bark extract (6.9 kg) (lot # RM052902-01) with total flavancontent of 80.4%; and excipient (5.0 kg of Candex) were combined toprovide a Univestin™ formulation (50.4 kg) having a blending ratio of85:15. Table 9 provides the quantification of the active Free-B-ringflavonoids and flavans of this specific batch of Univestin™(Lot#G1702-COX-2), determined using the methods provided in Examples 7and 9.

TABLE 11 Free-B-ring Flavonoid and Flavan Content of Univestin ™Formulation Active Components % Content 1. Flavonoids a. Baicalin 62.5% b. Minor Flavonoids i. Wogonin-7-glucuronide 6.7% ii. Oroxylin A7-glucuronide 2.0% iii. Baicalein 1.5% iv. Wogonin 1.1% v.Chrysin-7-glucuronide 0.8% vi. 5-Methyl-wogonin-7-glucuronide 0.5% vii.Scutellarin 0.3% viii. Norwogonin 0.3% ix. Chrysin <0.2%   x. Oroxylin A<0.2%   c. Total Free-B-ring Flavonoids 75.7%  2. Flavans a. Catechin9.9% b. Epicatechin 0.4% c. Subtotal Flavans 10.3%  3. Total ActiveIngredients  86%

With reference to Table 9, this specific batch of Univestin™ contains86% total active ingredients, including 75.7% Free-B-ring flavonoids and10.3% flavans. Two different dosage levels of final product in capsuleform were produced from this batch of Univestin™ (50.0 kg): 125 mg perdose (60 capsules) and 250 mg per dose (60 capsules). The final productwas evaluated in a human clinical trial as described in Example 15.

Using the same approach, two other batches of Univestin™ were preparedusing a combination of a standardized Free-B-ring flavonoid extract fromScutellaria baicalensis roots and a standardized flavan extract fromAcacia catechu bark having a blending ratio of 50:50 and 20:80,respectively.

Example 15 Measurements of Dose Response and IC₅₀ Values of COX EnzymeInhibitions from Three Formulations of Univestin™

The three different formulations of Univestin™ are produced as providedin Example 14 were tested for COX-1 and COX-2 inhibitory activity asdescribed in Example 6. All three formulation show significant doseresponse inhibition of COX enzyme activities as illustrated in FIGS. 11,12 and 13).

Example 16 Measurements of Dose Response and IC₅₀ Values of LO EnzymeInhibition from a Formulation of Univestin™

A Univestin™ sample was produced as outlined in Example 14, using acombination of a standardized Free-B-ring flavonoid extract fromScutellaria baicalensis roots and a standardized flavan extract fromAcacia catechu bark with a blending ratio of 80:20. The sample wastitrated in tissue culture media containing THP-1 or HT-29 cells;monocyte cell lines that express COX-1, COX-2 and 5-LO. A competitiveELISA for LTB₄ (LTB₄; Neogen, Inc., Cat#406110) was used to assess theeffect of Univestin™ on newly synthesized levels of LTB₄ present in eachcell line as a measure of Univestin™'s inhibitory effect on the 5-LOpathway. The assay was performed in duplicate by adding 160,000 to180,000 cells per well in 6-well plates. Univestin™ was added to theTHP-1 cultures at 3, 10, 30 and 100 μg/mL and incubated overnight(˜12-15 hrs) at 37° C. with 5% CO₂ in a humidified environment. Theresults are set forth in FIG. 14, which shows that the production ofnewly LPS-induced LTB₄ was almost completely inhibited by the additionof Univestin™ to the THP-1 cultures between 3 and 10 μg/mL.

Univestin™ and ibuprofen, another known 5-LO inhibitor, were added tothe HT-29 cells at 3 μg/mL and incubated 48 hrs at 37° C. with 5% CO₂ ina humidified environment. Each treated cell line was then harvested bycentrifugation and disrupted by gentle dounce homogenization lysis inphysiological buffers. As shown in FIG. 15, Univestin™ inhibitedgeneration of 80% of the newly synthesized LTB₄ in HT-29 cells.Ibuprofen only showed a 20% reduction in the amount of LTB₄ over thesame time period.

Example 17 Differential Inhibition of Cox-2 but not Cox-1 GeneExpression by Univestin™ Vs. Other NSAIDs

To evaluate whether Univestin™ is operating on the genomic level,isolated human, peripheral blood monocytes (PBMCs) were stimulated withlipopolysaccharide (LPS), treated with Univestin™ as illustrated inExample 14, celecoxib, ibuprofen or acetaminophen, and the total RNAproduced was then harvested and evaluated by semi-quantitative RT-qPCR.Specifically, the assay was constructed by adding 130,000 cells per wellin 6-well plates. The cells were then stimulated with 10 ng/mL LPS andco-incubated with Univestin™ at 1, 3, 10, 30 and 100 μg/mL andcelecoxib, ibuprofen and acetaminophen at 3 μg/mL for 18 hours at 37° C.with 5% CO₂ in a humidified environment. Each cell-treatment conditionwas then harvested by centrifugation and total RNA produced was isolatedusing TRIzol® reagent (Invitrogen™ Life Technologies, Cat#15596-026) andthe recommended TRIzol® reagent protocol. Total RNA was reversetranscribed using Moloney Murine Leukemia Virus reverse transcriptase(M-MLV RT; Promega Corp., Cat#M1701) using random hexamers (PromegaCorp., Cat#C1181). qPCR experiments were performed on an ABI Prism®7700Sequence Detection System using pre-developed validated Assays-on-Demandproducts (AOD from Applied Biosystems, Inc., Cat#4331182) for 18S rRNAinternal standard and gene specific assays. Gene specific expressionvalues were standardized to their respective 18S rRNA gene expressionvalues (internal control) and then the no-LPS no-drug treatmentcondition normalized to 100. Treatment conditions are relative to thisnull condition.

Univestin™ decreased normalized gene expression of cox-2 by over100-fold, while cox-1 normalized gene expression showed littlevariation. When PBMCs were treated with 3 μg/mL of Univestin™,celecoxib, ibuprofen or acetaminophen, only Univestin™ did not increasegene expression of cox-2. It is believed that this is the first reportof changes in gene expression levels of eicosinoids, cytokines,chemokines and other genes implicated in pain and inflammation pathwaysfollowing treatment with a mixture of Free-B-ring flavonoids and flavansusing semi-quantitative RT-qPCR techniques. This work has been coupledwork with ELISA-based assays to evaluate changes in protein levels aswell as enzyme function assays to evaluate alterations in proteinfunction. As a result of these studies, both genomic and proteomiccoupled effects following treatment with Univestin™ have beendemonstrated. Other studies cited in the literature have used proteinspecific methods to infer gene expression rather than show it directly.The results are set forth in FIGS. 16 and 17.

Example 18 Evaluation of the Efficacy of Univestin™ with In Vivo MouseEar Swelling Model

In order to test whether Univestin™ could be used to treat inflammationin vivo, the composition, prepared as described in Example 14, wasadministered by oral gavage to 4-5 week old ICR mice (Harlan Labs) oneday before treatment of their ears with AA. Test mice were fed doseequivalents of 50, 100 and 200 mg/kg of Univestin™ suspended in oliveoil while control mice were fed only olive oil. The following day, 20 μLof 330 mM AA in 95% alcohol was applied to one ear of each mouse, whilealcohol was applied to the other ear as a control. Mice treated withUnivestin™ showed a measurable dose response that tracked withincreasing doses of Univestin™, as demonstrated in FIG. 18. Withreference to FIG. 18, the 200 mg/kg dose reduces swelling by over 50% ascompared to the minus Univestin™ control. The 50 mg/kg dose ofUnivestin™ was as effective as the 50 mg/kg dose of another stronganti-inflammatory, indomethacin.

Example 19 Evaluation Efficacy of Univestin™ with In Vivo Mouse AnkleJoint Swelling Model

Since Univestin™ is designed to target joint pain, a solution of 20 μLof 100 mM AA in 95% ethanol was injected into the hind ankle joints of4-5 week old ICR mice (Harlan Labs) to generate swelling. The test groupwas fed 100 mg/kg of Univestin™ suspended in olive oil ˜12 hours beforewhile another group was not given Univestin™. Control groups includedmice that had not received arachidonic acid injections (negativecontrol) and a group that had 95% ethanol without AA injected (vehiclecontrol). These groups were also not given Univestin™. The results areset forth in FIG. 19. With reference to FIG. 19, the mice givenUnivestin™ that were injected with AA showed background levels ofswelling as compared to the controls and the untreated arachidonicinjected group. These results demonstrate the effectiveness ofUnivestin™ for reducing swelling in joints, the site of action.

Example 20 Clinical Evaluation of the Efficacy of Free-B-Ring Flavonoidsand Flavans on the Relief of Pain Caused by Rheumatoid Arthritis orOsteoarthritis of the Knee and/or Hip

This clinical study was a single-center, randomized, double-blind,placebo-controlled study. Sixty subjects (n=60) with rheumatoidarthritis or osteoarthritis of the knee and/or hip were randomly placedinto one of the following four groups:

A₀ Placebo n = 15 Placebo A₁ Dose 1 n = 15 Univestin ™ 250 mg/day (125mg b.i.d.) A₂ Dose 2 n = 15 Univestin ™ 500 mg/day (250 mg b.i.d.) A₃Active Control n = 15 Celecoxib 200 mg/day (100 mg b.i.d.)

The Univestin™ was prepared as described in Example 14. This specificbatch of Univestin™ (lot#G1702-COX-2) contains 86% total activeingredients, including 75.7% Free-B-ring flavonoids and 10.3% flavans.Celecoxib, also known as Celebrex™, is a trade name for a prescriptiondrug that is a COX-2 selective inhibitor.

Subjects were sex-matched and recruited from ages 40 to 75. Treatmentconsisted of oral administration for 90 days of the placebo or activecompound (Univestin™ or celecoxib) according to the above dose schedule.Subjects taking NSAIDs engaged in a two-week washout period beforebeginning the study. Physical activity was not restricted, nor were thesubjects given any advice as to diet. Subjects were free to withdrawfrom the trial at any time for any reason. The efficacy of thetreatments was evaluated at 30, 60 and 90 days of oral administration byphysicians, using the Western Ontario and McMaster Universities (WOMAC)Osteo-Arthritis Index (See Lingard et al. (2001) J. Bone & Joint Surg.83:1856-1864; Soderman and Malchau (2000) Acta Orthop. Scand.71(1):39-46). This protocol was reviewed and approved by an IRB boardfrom University of Montreal.

The WOMAC was administered to subjects preferably in the doctor'soffice. They were asked to read and respond to a questionnaire on theirown or via proxy in the waiting room of the doctor's office or wereinterviewed by project personnel over the telephone and the data weretranscribed in the computer database. This offered a stable environmentamong patients and reduced the possibility of bias due to different homeenvironments among patients. Between groups differences for allmeasurements were evaluated with One-Way Analysis of Variance andTukey's Least Significant Difference for multiple comparisons. Allquestions were assigned a weight from 0 to 4 depending on the severityof pain, stiffness or impaired function. These values were thenconverted to percentages normalized to 100 and reported as WOMAC scores.Higher values are indicative of greater impairment. Table 12 sets forththe mean WOMAC index scores for pain, stiffness and function for 250 mgand 500 mg per day Univestin™ compared to celecoxib at 200 mg per dayand the placebo before treatment (baseline) and at 30, 60 and 90 daysafter treatment. The lower the score, the less pain and stiffness andbetter function a patient has.

TABLE 12 WOMAC Index Scores at Baseline and at 30, 60 and 90 DaysUnivestin ™ Univestin ™ Celecoxib 250 500 200 Placebo WOMAC Std Std StdStd INDICE Mean Dev Mean Dev Mean Dev Mean Dev Pain-baseline 54.33 19.960.33 23.34 55 22.28 49.33 15.1 Pain-30 days 41.33 19.22 36 22.93 5023.09 41.67 15.55 Pain-60 days 40.71 16.62 40.77 19.77 30 16.46 57.3116.66 Pain-90 days 41.79 16.36 27.69 21.57 31.67 16.42 50 14.43Stiffness- 63.33 26.92 61.67 23.84 47.5 21.75 46.67 21.37 baselineStiffness-30 41.67 16.14 44.17 21.06 39.42 18.29 59.17 20.85 daysStiffness-60 37.5 18.99 39.42 19.66 37.5 29.76 46.15 24.68 daysStiffness-90 39.29 20.72 28.85 21.28 29.17 25.19 49.04 18.01 daysFunction- 58.41 22.74 62.92 17.68 49.38 10.33 52.82 8.29 baselineFunction-30 42.09 14.51 47.59 17.18 48.43 9.29 51.88 14.8 daysFunction-60 41.47 7.75 41.59 7.34 41.23 9.12 49.64 7.16 days Function-9042.44 17.08 38.12 13.21 44.41 11.06 50.95 12.73 days

Table 13 sets forth the mean absolute change in WOMAC scores for pain,stiffness and function. They are expressed as the difference between thebaseline and the scores given at 30, 60 and 90 days. The more negativethe score, the greater the improvement.

TABLE 13 Mean Absolute Change in WOMAC Scores at 30, 60 and 90 Days*Absolute Univestin ™ 250 Univestin ™ 500 Celecoxib 200 Placebo ChangeMean Std Dev Mean Std Dev Mean Std Dev Mean Std Dev Pain-30 days −1324.41 −24.33 18.7 −4.23 15.92 −7.67 26.98 Pain-60 days −14.64 26.85−17.31 35.27 −22.31 22.51 5 13.54 Pain-90 days −13.57 22.91 −30.38 21.06−16.67 21.36 −2.31 15.89 Stiffness-30 −21.67 24.31 −17.5 18.18 −8.6520.66 12.5 29.88 days Stiffness-60 −28.57 27.05 −21.15 33.61 −9.62 29.82−1.92 30.55 days Stiffness-90 −26.79 27.67 −31.73 20.17 −13.54 37.48−0.96 26.74 days Function-30 −16.32 19.58 −15.33 18.28 −0.37 6.86 −0.9414.05 days Function-60 −18.11 24.36 −21.4 19.79 −6.97 13.66 −3.49 11.81days Function-90 −17.13 23.69 −24.87 23.25 −2.78 8.34 −2.18 11.27 days*These data contain only subjects who completed the study.

It is very difficult to ascribe a standard deviation to a group mean ina clinic trial due to the severe differences that appear in the data.Rather, confidence limits for the mean are preferred because they give alower and upper limit for the mean and the narrower the interval, themore precise the estimate of the mean. Confidence limits are expressedin terms of a confidence coefficient. A 95% confidence interval is themost commonly used interval to describe a mean in this type ofstatistical analysis. This does not imply that there is a 95%probability that the interval contains the true mean. Instead, the levelof confidence is associated with the method of calculating the interval.The confidence coefficient is simply the proportion of samples of agiven size that may be expected to contain the true mean. That is, for a95% confidence interval, if many samples are collected and theconfidence interval computed, in the long run about 95% of theseintervals would contain the true mean. With this in mind, the 95%confidence interval was computed for the WOMAC scores for pain,stiffness and function at 30, 60 and 90 days.

Raw/non standardized scores for the WOMAC scores based on a five pointLikert scale with a range between 1 and 5 were chosen to represent thefinal pain, stiffness and impaired function indices (FIGS. 20-31).Standardization to a scale between 0 and 100 was used in other sectionsfor uniformity (see Tables 12 and 13) and to enhance the appreciation ofthe magnitudes of changes. However, given that all the figures are basedon the same 1-5 point scales the raw data were plotted since they moreaccurately reflect the methods by which these scores were obtained fromthe patient questionnaires. In other words, since the patients weregiven a choice between 1 and 5 these representations better reflect thepatient's response as opposed to the standardized or transformed scoreof 0-100 that does not reflect the patient's perception of possiblerange of answers.

Clear trends exist showing that for the pain indice that Univestin™ at250 and 500 mg/day reduced pain over the 90 day treatment period basedon the patient responses. Celecoxib also reduces pain over this sameperiod of time compared to the placebo, which does not. However,celecoxib does not seem to be as effective as Univestin™ at both dosagesin reducing stiffness, since the confidence intervals heavily overlappedthose of the placebo. Finally, Univestin™ at both doses clearly improvedfunctional impairment, but celecoxib does not compared to placebo. Thegraphic representations contain all subjects even if they did notcomplete the study. Each confidence interval, however, is valid based onthe number of subjects that were present at the time the WOMAC testswere taken so the trends still hold. These data are plotted in FIGS. 20through 31.

Example 21 Clinical Evaluation of the Efficacy of Free-B-Ring Flavonoidsand Flavans on BMI and Weight Loss Due to an Increase in Function

Additional measurements taken during the clinical trial were height andweight. All subjects in all groups (see Example 20) were measured forheight and weight at 30 and 90 days of treatment. The subjects weregiven no recommendations on diet or exercise in order not to bias theresults toward reduction of BMI and weight loss. Table 14 illustratesthe changes in weight and BMI that occurred after treatment for 30 and90 days.

TABLE 14 Change in Mean Weight (kg) and BMI (kg/m²) at 30 and 90 DaysGroup Univestin ™ Univestin ™ Celecoxib 250 500 200 Placebo Std Std StdStd Mean Dev Mean Dev Mean Dev Mean Dev Weight-30 days −3.60 3.76 −2.403.31 −2.00 3.08 −.60 1.99 Weight-90 days −5.36 3.43 −4.15 4.81 −3.174.88 −.08 1.50 BMI-30 days −1.28 1.33 −.80 1.13 −.68 1.06 −.20 .64BMI-90 days −1.84 1.14 −1.39 1.64 −1.07 1.67 −.02 .54

Based on these data, the 250 mg/day dose of Univestin™ gave the greatestamount of weight loss and change in BMI followed by the 500 mg/day doseof Univestin™ and then celecoxib. The placebo had no effect on weight orBMI.

It is not believed that there are any other reports in the literature ofanti-inflammatory compounds being used to effect weight loss or changesin BMI. Though the subjects were given no advice on exercise, thegreater functional capabilities gained after treatment, especially withUnivestin™, may have allowed them to exercise more on their own accord.Alternatively, Univestin™ may be increasing thermogenesis, lipolysis, orcausing an under utilization of carbohydrates or fat in the diet. FIGS.32 and 33 illustrate the BMI and weight loss seen for Univestin™ after30 and 90 days of treatment.

Example 22 Clinical Evaluation of the Efficacy of Free-B-Ring Flavonoidsand Flavans on Lowering of Blood Glucose Due to an Increase in Function

Blood glucose was also taken at 0 (baseline), 30 days and 90 days aftertreatment (see Example 20). These measurements were reported in mmoleper liter. The data is also shown in mg/dL. Table 15 sets forth bloodglucose levels after 30 and 90 days of treatment with Univestin™ at 250and 500 mg/day.

TABLE 15 Change in Blood Glucose after 30 and 90 days of Treatment GroupUnivestin ™ 250 Univestin ™ 500 Placebo Std Std Std mmol/L mg/dL Devmmol/L mg/dL Dev mmol/L mg/dL Dev Glucose- 5.24 94.32 .74 5.09 91.62 .674.82 86.76 .80 Baseline Glucose- 5.10 91.80 .71 4.75 85.50 .55 5.0891.44 .54 30 days Glucose- 4.88 87.84 .72 4034 78.12 .36 4.71 84.78 .5690 days Percent −7.52 −12.79 .94 Change By 90 days

These data suggest that both the 250 and the 500 mg/day doses ofUnivestin™ are significantly lowering blood glucose levels over time.This impact may or may not be related to the loss of weight observedabove or to the presumed increase in activity as functional impairmentimproved. It may also be possible that Univestin™ is acting directly toimprove glucose metabolism by decreasing glucose tolerance or byutilizing carbohydrates more effectively.

The invention claimed is:
 1. A composition comprised of a mixture of anextract derived from Scutellaria enriched for Free-B-ring flavonoidscontaining baicalin and an extract derived from Acacia enriched forflavans containing catechin or epicatechin, wherein the ratio ofFree-B-ring flavonoid:flavan in the composition is about 85:15.
 2. Thecomposition of claim 1, wherein at least 80% of the total weight of thecomposition comprises Free-B-ring flavonoids and flavans.
 3. Thecomposition of claim 1, wherein the major active components of themixture are the Free-B-ring flavonoids and the flavans.
 4. Thecomposition of claim 1, wherein the major active ingredients of themixture are baicalin, catechin, and epicatechin.
 5. The composition ofclaim 1, wherein the Free-B-ring flavonoids and the flavans are enrichedfrom a plant part selected from the group consisting of stems, stembarks, trunks, trunk barks, twigs, tubers, roots, root barks, youngshoots, seeds, rhizomes, flowers and other reproductive organs, leavesand other aerial parts.
 6. The composition of claim 1, wherein theFree-B-ring flavonoids are enriched from Scutellaria baicalensis orScutellaria orthocalyx.
 7. The composition claim 1, wherein said flavansare enriched from a plant species selected from the group consisting ofthe Acacia catechu, Acacia concinna, Acacia farnesiana, Acacia Senegal,Acacia speciosa, Acacia arabica, A. caesia, A. pennata, A. sinuata. A.mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A. holosereciaand A. mangium.
 8. The composition of claim 1, wherein the Free-B-ringflavonoids are enriched from Scutellaria baicalensis and the flavans areenriched from Acacia catechu.
 9. The composition of claim 1, furthercomprising an adjuvant, excipient or carrier.
 10. The composition ofclaim 9, wherein the adjuvant, excipient or carrier is selected from oneor more of calcium-based salts, silica, boron, histidine, glucosaminesulfate, chondroitin sulfate, copper gluconate, cellulose, dextrose,saline, water, oil, vitamin D, and shark and bovine cartilage.